Methanotrophic carbon metabolism pathway genes and enzymes

ABSTRACT

Genes have been isolated from a Methylomonas sp encoding enzymes in the carbon flux pathway. The genes encode a 2-keto-3-deoxy-6-phosphogluconate (KDPGA) and a fructose bisphosphate aldolase (FFBPA), as well as numerous other genes. The genes will be useful in C1 metabolizing microorganisms for the manipulation of the carbon flux pathway.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/229,906, filed Sep. 1, 2000.

FIELD OF THE INVENTION

[0002] The invention relates to the field of molecular biology andmicrobiology. More specifically, the invention relates to genes involvedin the conversion of hexose sugars into 3-carbon metabolites inmethanotrophic bacteria.

BACKGROUND OF THE INVENTION

[0003] Methanotrophic bacteria are defined by their ability to usemethane as their sole source of carbon and energy. Although methanol isan obligate intermediate in the oxidation of methane, the ability togrow on methanol alone is highly variable among the obligatemethanotrophs (Green, Peter. Taxonomy of Methylotrophic Bacteria. In:Methane and Methanol Utilizers (Biotechnology Handbooks 5) J. ColinMurrell and Howard Dalton eds. 1992 Pleanum Press NY. pp. 23-84)). Theconversion of C1 compounds to complex molecules with C-C bonds isdifficult and expensive by traditional chemical synthetic routes.Traditionally, methane is first converted to synthesis gas which is thenused to produce other small molecular weight industrial precursors. Thebasic problem is activation of the methane molecule, a process which isthermodynamically very difficult to achieve by chemical means.Methanotrophs have proved useful mediators of this problem.

[0004] Methane monooxygenase is the enzyme required for the primary stepin methane activation and the product of this reaction is methanol(Murrell et al., Arch. Microbiol. (2000), 173(5-6), 325-332). Thisremarkable reaction occurs at ambient temperatures and pressures,whereas chemical transformation of methane to methanol requirestemperatures of hundreds of degrees and high pressures (Grigoryan, E.A., Kinet. Catal. (1999), 40(3), 350-363; WO 2000007718; U.S. Pat. No.5,750,821). It is this ability to transform methane under ambientconditions, along with the abundance of methane, that makes thebiotransformation of methane a potentially unique and valuable process.

[0005] The commercial applications of biotransformation of methane havehistorically fallen broadly into three categories: 1) Production ofsingle cell protein, (Villadsen, John, Recent Trends Chem. React. Eng.,[Proc. Int. Chem. React. Eng. Conf.], 2nd (1987), Volume 2, 320-33.Editor(s): Kulkarni, B. D.; Mashelkar, R. A.; Sharma, M. M. Publisher:Wiley East, New Delhi, India; Naguib, M., Proc. OAPEC Symp.Petroprotein, [Pap.] (1980), Meeting Date 1979, 253-77 Publisher: Organ.Arab Pet. Exporting Countries, Kuwait, Kuwait); 2) epoxidation ofalkenes for production of chemicals (U.S. Pat. No. 4,348,476); and 3)biodegradation of chlorinated pollutants (Tsien et al., Gas, Oil, Coal,Environ. Biotechnol. 2, [Pap. Int. IGT Symp. Gas, Oil, Coal, Environ.Biotechnol.], 2nd (1990), 83-104, Editor(s): Akin, Cavit; Smith, Jared.Publisher: Inst. Gas Technol., Chicago, Ill.; WO 9633821; Merkley etal., Biorem. Recalcitrant Org., [Pap. Int. In Situ On-Site Bioreclam.Symp.], 3rd (1995), 165-74. Editor(s): Hinchee, Robert E; Anderson,Daniel B.; Hoeppel, Ronald E. Publisher: Battelle Press, Columbus, Ohio;Meyer et al., Microb. Releases (1993), 2(1), 11-22). Only epoxidation ofalkenes has experienced little commercial success due to low productyields, toxicity of products and the large amount of cell mass requiredto generate product.

[0006] Methanotrophic cells can further build the oxidation products ofmethane (i.e. formaldehyde) into more complex molecules such as protein,carbohydrate and lipids. For example, under certain conditionsmethanotrophs are known to produce exopolysaccharides (Ivanova et al.,Mikrobiologiya (1988), 57(4), 600-5); Kilbane, John J., II Gas, Oil,Coal, Environ. Biotechnol. 3, [Pap. IGT's Int. Symp.], 3rd (1991),Meeting Date 1990, 207-26. Editor(s): Akin, Cavit; Smith, Jared.Publisher: IGT, Chicago, Ill.). Similarly, methanotrophs are known toaccumulate both isoprenoid compounds and carotenoid pigments of variouscarbon lengths (Urakami et al., J. Gen. Appl. Microbiol. (1986), 32(4),317-41). Although these compounds have been identified in methanotrophs,they have not been microbial platforms of choice for production becausethese organisms have very poorly developed genetic systems, therebylimiting metabolic engineering ability for chemicals.

[0007] A necessary prerequisite to metabolic engineering ofmethanotrophs is a full understanding, and optimization, of the carbonmetabolism for maximum growth and/or product yield. In methanotrophicbacteria, methane is converted to biomolecules via a cyclic set ofreactions known as the ribulose monophosphate pathway (RuMP) cycle. TheRuMP pathway is comprised of three phases, each phase being a series ofenzymatic steps. The first phase (fixation) is the aldol condensation ofthree molecules of C-1 (formaldehyde) with three molecules of pentose(ribulose-5-phospate) to form three molecules of a six-carbon sugar(fructose-6-phosphate) catalyzed by hexulose monophosphate synthase.This fixation phase is common to all methylotrophic bacteria using theRuMP pathway.

[0008] The second phase is termed “cleavage” and results in splitting ofthat 6-carbon sugar into two 3-carbon molecules. This may be achievedvia two possible routes. Fructose-6-phosphate is either converted intofructose-1,6-biphosphate (FBP) by phosphofructokinase, and subsquentlycleaved by FBP aldolase (FBPA) to 3-carbon molecules, or oxidized to2-keto-3-deoxy-6-phosphogluconate (KDPG) and ultimately cleaved to3-carbon sugars by the enzyme catalyzed by KDPG aldolase. One of those3-carbon molecules is recycled back through the RuMP pathway and theother 3-carbon fragment is utilized for cell growth.

[0009] In the third phase (the “rearrangement” phase), the regenerationof 3 molecules of ribulose-5-phosphate is accomplished from the tworemaining molecules of fructose-6-phosphate (from stage 1) and the onemolecule of the 3-carbon sugar from stage 2. There are two possibleroutes to achieve the rearrangement. These routes in the rearrangementphase differ in that they involve either transaldolase (TA) orsedoheptulose-1,7-bisphosphatase (SBPase).

[0010] In methanotrophs and methylotrophs, the RuMP pathway may occur asone of three variants. These are the KDPGA/TA, FBPA/SBPase and FBPA/TApathways. However, only two of these variants are commonly found. Thesetwo pathways are the FBPA/TA (fructose bisphophotasealdolase/Transaldolase) or the KDPGA/TA (keto deoxy phosphgogluconatealdolase/transaldolase) pathway, wherein only the FBPA/TA pathway isexergonic (Dijkhuizen et al. (1992) The Physiology and biochemistry ofaerobic methanol-utilizing gram negative and gram positive bacteria. In:Methane and Methanol utilizers. P. 149-Coin Murrell and Howard Dalton,Plenum Press NY). Available literature suggests that obligatorymethanotrophic bacteria such as Methylomonas rely solely on the KDPGA/TApathway (Entner-Douderoff Pathway), while facultative methylotrophsutilize either the FBPA/SBPase or the FBPA/TA pathway (Dijkhuizen et al.supra). Energetically, this pathway is not as efficient as theEmbden-Meyerhof pathway and thus could result in lower cellularproduction yields, as compared to organisms that do use the latterpathway. Therefore, a more energy efficient carbon processing pathwaywould greatly enhance the commercial viability of the methanotrophicplatform for the generation of materials.

[0011] The problem to be solved therefore is to discover genes encodinga more energetically efficient carbon flux pathway that would enable amethanotrophic bacterial strain to better able to serve as a platformfor the production of proteins and carbon containing materials.Applicants have solved the stated problem by providing the genesencoding the carbon flux pathway in a strain of Methylomonas. Thispathway contains not only the expected elements of the Entner-DouderoffPathway (including the 2-keto- 3-deoxy-6-phosphogluconate aldolase) butadditionally contains the elements of the more energy efficientEmbden-Meyerhof pathway, containing the fructose-1,6-biphosphatealdolase. This discovery will permit the engineering of methanotrophsand other organisms for the energy efficient conversion of single carbonsubstrates such as methane and methanol to commercially useful productsin the food and feed and materials industries.

SUMMARY OF THE INVENTION

[0012] The present invention provides an isolated nucleic acid moleculeencoding a Methylomonas sp carbon flux enzyme, selected from the groupconsisting of:

[0013] (a) an isolated nucleic acid molecule encoding the amino acidsequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8,10, 12, 14, 16, 18, and 20;

[0014] (b) an isolated nucleic acid molecule that hybridizes with (a)under the following hybridization conditions: 0.1×SSC, 0.1% SDS, 65° C.and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS; and

[0015] (c) an isolated nucleic acid molecule that is complementary to(a) or (b).

[0016] Additionally the invention provides the gene products, encoded bythe present invention and chimera made from the instant genes byoperably linking the instant genes to suitable regulatory sequences.Similarly the invention provides transformed host cells expressing theinstant genes or their chimera.

[0017] The invention additionally provides a method of obtaining anucleic acid fragment encoding a carbon flux enzyme comprising:

[0018] (a) probing a genomic library with the nucleic acid fragment ofthe present invention;

[0019] (b) identifying a DNA clone that hybridizes with the nucleic acidfragment of the present invention; and

[0020] (c) sequencing the genomic fragment that comprises the cloneidentified in step (b),

[0021] wherein the sequenced genomic fragment encodes a carbon fluxenzyme.

[0022] Alternatively the invention provides a method of obtaining anucleic acid fragment encoding a carbon flux enzyme comprising:

[0023] (a) synthesizing at least one oligonucleotide primercorresponding to a portion of the sequence selected from the groupconsisting of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17, and 19;

[0024] (b) amplifying an insert present in a cloning vector using theoligonucleotide primer of step (a);

[0025] wherein the amplified insert encodes a portion of an amino acidsequence encoding a carbon flux enzyme.

[0026] In another embodiment the invention provides a method of alteringcarbon flow through a methanotrophic bacteria comprising,over-expressing at least one carbon flux gene selected from the groupconsisting of SEQ ID NO:1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 in amethanotrophic strain such that the carbon flow is altered through thestrain.

[0027] Additionally the invention provides a mutated gene encoding acarbon flux enzyme having an altered biological activity produced by amethod comprising the steps of:

[0028] (i) digesting a mixture of nucleotide sequences with restrictionendonucleases wherein said mixture comprises:

[0029] a) a native carbon flux gene;

[0030] b) a first population of nucleotide fragments which willhybridize to said native carbon flux gene;

[0031] c) a second population of nucleotide fragments which will nothybridize to said native carbon flux gene;

[0032] wherein a mixture of restriction fragments are produced;

[0033] (ii) denaturing said mixture of restriction fragments;

[0034] (iii) incubating the denatured said mixture of restrictionfragments of step (ii) with a polymerase;

[0035] (iv) repeating steps (ii) and (iii) wherein a mutated carbon fluxgene is produced encoding a protein having an altered biologicalactivity.

BRIEF DESCRIPTION OF THE DRAWINGS, SEQUENCE DESCRIPTIONS AND BIOLOGICALDEPOSITS

[0036]FIG. 1 is a schematic showing the enzyme catalyzed reaction of theEmbden-Meyerhof and the Entner-Douderoff carbon pathways present in theMethylomonas 16a strain.

[0037] The invention can be more fully understood from the followingdetailed description and the accompanying sequence descriptions whichform a part of this application.

[0038] The following sequence descriptions and sequences listingsattached hereto comply with the rules governing nucleotide and/or aminoacid sequence disclosures in patent applications as set forth in 37C.F.R. §1.821-1.825. The Sequence Descriptions contain the one lettercode for nucleotide sequence characters and the three letter codes foramino acids as defined in conformity with the IUPAC-IYUB standardsdescribed in Nucleic Acids Research 13:3021-3030 (1985) and in theBiochemical Journal 219 (No. 2), 345-373 (1984) which are hereinincorporated by reference. The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822. SEQ ID Nucleic SEQ ID Description acid PeptideTransaldolase: Carbon Flux 1 2 Transaldolase: Carbon Flux 3 4 Fructosebisphosphate 5 6 aldoslase: Carbon Flux Fructose bisphosphate 7 8aldoslase: Carbon Flux KHG/KDPG Aldolase: Carbon Flux 9 10Phosphoglucomutase: carbon Flux 11 12 Glucose 6 phosphate 13 14isomerase: Carbon flux Phosphofructokinase pyrophosphate 15 16dependent: Carbon Flux 6-Phosphogluconate 17 18 dehydratase: Carbon fluxGlucose 6 phosphate 1 19 20 dehydrogenase: Carbon Flux

[0039] Applicants made the following biological deposits under the termsof the Budapest Treaty on the International Recognition of the Depositof Micro-organisms for the Purposes of Patent Procedure: InternationalDepositor Identification Depository Reference Designation Date ofDeposit Methylomonas 16a ATCC PTA 2402 August 21 2000

DETAILED DESCRIPTION OF THE INVENTION

[0040] The invention relates to genes encoding enzymes in the carbonflux pathway from a methanotrophic bacteria. The pathway contains genesencoding fructose-1,6-biphosphate aldolase (FBP aldolase) and apyrophosphate dependent phosphofructokinase pyrophosphate which areindicative of the Embden-Meyerhof pathway typically not found inmethanotrophs. The Embden-Meyerhof pathway is energetically morefavorable than the carbon flux pathway typically associated with theseorganisms. Additionally the invention provides genes encoding elementsof the Entner-Douderoff Pathway, which is typically found inmethanotrophic bacteria. These genes include 6-Phosphogluconatedehydratase, a glucose-6-phosphate-1-dehydrogenase, and a2-keto-3-deoxy-6-phosphogluconate aldolase. Common to both pathways arenew genes encoding a transaldolase and a phosphoglucomutase. Knowledgeof the sequence of the present genes will be useful for altering thecarbon flow in methanotrophs and other bacteria resulting in moreproductive bacterial fermentation platforms for the production ofchemicals and food and feed products.

[0041] In this disclosure, a number of terms and abbreviations are used.The following definitions are provided. “Open reading frame” isabbreviated ORF. “Polymerase chain reaction” is abbreviated PCR.

[0042] The term “a C1 carbon substrate” refers to any carbon-containingmolecule that lacks a carbon-carbon bond. Examples are methane,methanol, formaldehyde, formic acid, methylated amines, methylatedthiols.

[0043] The term “RuMP” is the abbreviation for ribulose monophosphateand the “RuMP pathway” refers to the set of enzymes found inmethanotrophic bacteria responsible of the conversion of the methanemonooxygenase product (methanol, formaldehyde) to three carbon moietiesuseful for energy production in the methanotroph.

[0044] The term “Embden-Meyerhof pathway” refers to the series ofbiochemical reactions for conversion of hexoses such as glucose andfructose to important cellular 3-carbon intermediates such asglyceraldehyde 3 phosphate, dihydroxyacetone phosphate, phosphoenolpyruvate and pyruvate. These reactions typically proceed with net yieldof biochemically useful energy in the form of ATP. The key enzymesunique to the Embden-Meyerhof pathway are the phosphofructokinase andfructose 1,6 bisphosphate aldolase.

[0045] The term “Entner-Douderoff pathway” refers to a series ofbiochemical reactions for conversion of hexoses such as as glucose orfructose to the important 3-carbon cellular intermediates pyruvate andglyceraldehyde 3 phosphate, without any net production of biochemicallyuseful energy. The key enzymes unique to the Entner-Douderoff pathwayare the 6 phosphogluconate dehydratase and the ketodeoxyphosphogluconatealdolase.

[0046] The term “high growth methanotrophic bacterial strain” refers toa bacterium capable of growth with methane or methanol as the solecarbon and energy source and which possesses a functionalEmbden-Meyerhof carbon flux pathway resulting in yield of cell mass pergram of C1 substrate metabolized. The specific “high growthmethanotrophic bacterial strain” described herein is referred to as“Methylomonas 16a” or “16a”, which terms are used interchangeably.

[0047] The term “methanotroph” or “methanotrophic bacteria” will referto a prokaryotic microorganism capable of utilizing methane as itsprimary carbon and energy source.

[0048] As used herein, an “isolated nucleic acid fragment” is a polymerof RNA or DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA.

[0049] The term “carbon flux gene” will refer to any gene encoding anenzyme that functions to convert C1 substrates in methanotrophicbacteria to metabolically useful products. As used herein “carbon fluxgenes” will be those encoding a phosphoglucomutase, a transaldolase, aglucose-6-phosphate isomerase, a phosphofructokinase (pyrophosphatedependent), a 6-Phosphogluconate dehydratase, and a glucose 6 phosphate1 dehydrogenase, as well as the distinctive fructose bisphosphatealdolase and keto deoxy phosphogluconate aldolase.

[0050] “Carbon Flux enzymes” will refer to the gene products of thecarbon flux genes.

[0051] The term “transaldolase ” will be abbrevaited “TA” and will referto an enzyme that catalyzes the reaction of sedoheptulose 7-phosphateand D-glyceraldehyde 3-phosphate to give D-erythrose 4-phosphate andD-fructose 6-phosphate

[0052] The term “fructose bisphosphate aldolase” will be abbreviated“FFBPA” and will refer to an enzyme that catalyzes the reaction ofD-fructose 1,6-bisphosphate to give glycerone- phosphate andD-glyceraldehyde 3-phosphate.

[0053] The term “keto deoxy phosphogluconate aldolase” will beabbreviated “KDPGA” and will refer to an enzyme that catalyzes thereaction of 2-dehydro-3-deoxy-d-gluconate 6-phosphate to give pyruvateand D-glyceraldehyde 3-phosphate.

[0054] The term “phosphoglucomutase ” and will refer to an enzyme thatcatalyzes the interconversion of glucose-6-phosphate toglucose-1-phosphate.

[0055] The term “glucose-6-phosphate isomerase” and will refer to anenzyme that catalyzes the conversion of fructose-6-phosphate toglucose-6-phosphate.

[0056] The term “phosphofructokinase” and will refer to an enzyme thatcatalyzes the conversion of fructose-6-phosphate tofructose-1,6-bisphosphate.

[0057] The term “6-phosphogluconate dehydratase” and will refer to anenzyme that catalyzes the conversion of 6-phosphogluconate to2-keto-3-deoxy-6-phosphogluconate (KDPG).

[0058] The term “6-phosphogluconate-6-phosphate-1 dehydrogenase” andwill refer to an enzyme that catalyzes the conversion ofglucose-6-phosphate to 6-phosphogluconate.

[0059] As used herein, “substantially similar” refers to nucleic acidfragments wherein changes in one or more nucleotide bases results insubstitution of one or more amino acids, but do not affect thefunctional properties of the protein encoded by the DNA sequence.“Substantially similar” also refers to nucleic acid fragments whereinchanges in one or more nucleotide bases does not affect the ability ofthe nucleic acid fragment to mediate alteration of gene expression byantisense or co-suppression technology. “Substantially similar” alsorefers to modifications of the nucleic acid fragments of the instantinvention such as deletion or insertion of one or more nucleotide basesthat do not substantially affect the functional properties of theresulting transcript. It is therefore understood that the inventionencompasses more than the specific exemplary sequences.

[0060] For example, it is well known in the art that alterations in agene which result in the production of a chemically equivalent aminoacid at a given site, but do not effect the functional properties of theencoded protein are common. For the purposes of the present inventionsubstitutions are defined as exchanges within one of the following fivegroups:

[0061] 1. Small aliphatic, nonpolar or slightly polar residues: Ala,Ser, Thr (Pro, Gly);

[0062] 2. Polar, negatively charged residues and their amides: Asp, Asn,Glu, Gln;

[0063] 3. Polar, positively charged residues: His, Arg, Lys;

[0064] 4. Large aliphatic, nonpolar residues: Met, Leu, lie, Val (Cys);and

[0065] 5. Large aromatic residues: Phe, Tyr, Trp.

[0066] Thus, a codon for the amino acid alanine, a hydrophobic aminoacid, may be substituted by a codon encoding another less hydrophobicresidue (such as glycine) or a more hydrophobic residue (such as valine,leucine, or isoleucine). Similarly, changes which result in substitutionof one negatively charged residue for another (such as aspartic acid forglutamic acid) or one positively charged residue for another (such aslysine for arginine) can also be expected to produce a functionallyequivalent product.

[0067] In many cases, nucleotide changes which result in alteration ofthe N-terminal and C-terminal portions of the protein molecule wouldalso not be expected to alter the activity of the protein.

[0068] Each of the proposed modifications is well within the routineskill in the art, as is determination of retention of biologicalactivity of the encoded products. Moreover, the skilled artisanrecognizes that substantially similar sequences encompassed by thisinvention are also defined by their ability to hybridize, understringent conditions (0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC,0.1% SDS followed by 0.1×SSC, 0.1% SDS), with the sequences exemplifiedherein. Preferred substantially similar nucleic acid fragments of theinstant invention are those nucleic acid fragments whose DNA sequencesare at least 80% identical to the DNA sequence of the nucleic acidfragments reported herein. More preferred nucleic acid fragments are atleast 90% identical to the DNA sequence of the nucleic acid fragmentsreported herein. Most preferred are nucleic acid fragments that are atleast 95% identical to the DNA sequence of the nucleic acid fragmentsreported herein.

[0069] A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength. Hybridization and washing conditions are well known andexemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 andTable 11.1 therein (entirely incorporated herein by reference). Theconditions of temperature and ionic strength determine the “stringency”of the hybridization. Stringency conditions can be adjusted to screenfor moderately similar fragments, such as homologous sequences fromdistantly related organisms, to highly similar fragments, such as genesthat duplicate functional enzymes from closely related organisms.Post-hybridization washes determine stringency conditions. One set ofpreferred conditions uses a series of washes starting with 6×SSC, 0.5%SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDSat 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at50° C. for 30 min. A more preferred set of stringent conditions useshigher temperatures in which the washes are identical to those aboveexcept for the temperature of the final two 30 min washes in 0.2×SSC,0.5% SDS was increased to 60° C. Another preferred set of highlystringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65°C. Hybridization requires that the two nucleic acids containcomplementary sequences, although depending on the stringency of thehybridization, mismatches between bases are possible. The appropriatestringency for hybridizing nucleic acids depends on the length of thenucleic acids and the degree of complementation, variables well known inthe art. The greater the degree of similarity or homology between twonucleotide sequences, the greater the value of Tm for hybrids of nucleicacids having those sequences. The relative stability (corresponding tohigher Tm) of nucleic acid hybridizations decreases in the followingorder: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100nucleotides in length, equations for calculating Tm have been derived(see Sambrook et al., supra, 9.50-9.51). For hybridizations with shorternucleic acids, i.e., oligonucleotides, the position of mismatchesbecomes more important, and the length of the oligonucleotide determinesits specificity (see Sambrook et al., supra, 11.7-11.8). In oneembodiment the length for a hybridizable nucleic acid is at least about10 nucleotides. Preferable a minimum length for a hybridizable nucleicacid is at least about 15 nucleotides; more preferably at least about 20nucleotides; and most preferably the length is at least 30 nucleotides.Furthermore, the skilled artisan will recognize that the temperature andwash solution salt concentration may be adjusted as necessary accordingto factors such as length of the probe.

[0070] A “substantial portion” of an amino acid or nucleotide sequencecomprising enough of the amino acid sequence of a polypeptide or thenucleotide sequence of a gene to putatively identify that polypeptide orgene, either by manual evaluation of the sequence by one skilled in theart, or by computer-automated sequence comparison and identificationusing algorithms such as BLAST (Basic Local Alignment Search Tool;Altschul, S. F., et al., (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or morecontiguous amino acids or thirty or more nucleotides is necessary inorder to putatively identify a polypeptide or nucleic acid sequence ashomologous to a known protein or gene. Moreover, with respect tonucleotide sequences, gene specific oligonucleotide probes comprising20-30 contiguous nucleotides may be used in sequence-dependent methodsof gene identification (e.g., Southern hybridization) and isolation(e.g., in situ hybridization of bacterial colonies or bacteriophageplaques). In addition, short oligonucleotides of 12-15 bases may be usedas amplification primers in PCR in order to obtain a particular nucleicacid fragment comprising the primers. Accordingly, a “substantialportion” of a nucleotide sequence comprises enough of the sequence tospecifically identify and/or isolate a nucleic acid fragment comprisingthe sequence. The instant specification teaches partial or completeamino acid and nucleotide sequences encoding one or more particularmicrobial proteins. The skilled artisan, having the benefit of thesequences as reported herein, may now use all or a substantial portionof the disclosed sequences for purposes known to those skilled in thisart. Accordingly, the instant invention comprises the complete sequencesas reported in the accompanying Sequence Listing, as well as substantialportions of those sequences as defined above.

[0071] The term “complementary” is used to describe the relationshipbetween nucleotide bases that are capable to hybridizing to one another.For example, with respect to DNA, adenosine is complementary to thymineand cytosine is complementary to guanine. Accordingly, the instantinvention also includes isolated nucleic acid fragments that arecomplementary to the complete sequences as reported in the accompanyingSequence Listing as well as those substantially similar nucleic acidsequences.

[0072] The term “percent identity”, as known in the art, is arelationship between two or more polypeptide sequences or two or morepolynucleotide sequences, as determined by comparing the sequences. Inthe art, “identity” also means the degree of sequence relatednessbetween polypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, New York (1988);Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.)Academic Press, New York (1993); Computer Analysis of Sequence Data,Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NewJersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G.,ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M.and Devereux, J., eds.) Stockton Press, NY (1991). Preferred methods todetermine identity are designed to give the best match between thesequences tested. Methods to determine identity and similarity arecodified in publicly available computer programs. Sequence alignmentsand percent identity calculations may be performed using the Megalignprogram of the LASERGENE bioinformatics computing suite (DNASTAR Inc.,Madison, Wis.). Multiple alignment of the sequences was performed usingthe Clustal method of alignment (Higgins and Sharp (1989) CABIOS.5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTHPENALTY=10). Default parameters for pairwise alignments using theClustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALSSAVED=5.

[0073] Suitable nucleic acid fragments (isolated polynucleotides of thepresent invention) encode polypeptides that are at least about 70%identical, preferably at least about 80% identical to the amino acidsequences reported herein. Preferred nucleic acid fragments encode aminoacid sequences that are about 85% identical to the amino acid sequencesreported herein. More preferred nucleic acid fragments encode amino acidsequences that are at least about 90% identical to the amino acidsequences reported herein. Most preferred are nucleic acid fragmentsthat encode amino acid sequences that are at least about 95% identicalto the amino acid sequences reported herein. Suitable nucleic acidfragments not only have the above homologies but typically encode apolypeptide having at least 50 amino acids, preferably at least 100amino acids, more preferably at least 150 amino acids, still morepreferably at least 200 amino acids, and most preferably at least 250amino acids.

[0074] “Codon degeneracy” refers to the nature in the genetic codepermitting variation of the nucleotide sequence without effecting theamino acid sequence of an encoded polypeptide. Accordingly, the instantinvention relates to any nucleic acid fragment that encodes all or asubstantial portion of the amino acid sequence encoding the instantmicrobial polypeptides as set forth in SEQ ID NOs:2, 4, 6, 8, and 10.The skilled artisan is well aware of the “codon-bias” exhibited by aspecific host cell in usage of nucleotide codons to specify a givenamino acid. Therefore, when synthesizing a gene for improved expressionin a host cell, it is desirable to design the gene such that itsfrequency of codon usage approaches the frequency of preferred codonusage of the host cell.

[0075] “Synthetic genes” can be assembled from oligonucleotide buildingblocks that are chemically synthesized using procedures known to thoseskilled in the art. These building blocks are ligated and annealed toform gene segments which are then enzymatically assembled to constructthe entire gene. “Chemically synthesized”, as related to a sequence ofDNA, means that the component nucleotides were assembled in vitro.Manual chemical synthesis of DNA may be accomplished usingwell-established procedures, or automated chemical synthesis can beperformed using one of a number of commercially available machines.Accordingly, the genes can be tailored for optimal gene expression basedon optimization of nucleotide sequence to reflect the codon bias of thehost cell. The skilled artisan appreciates the likelihood of successfulgene expression if codon usage is biased towards those codons favored bythe host. Determination of preferred codons can be based on a survey ofgenes derived from the host cell where sequence information isavailable.

[0076] “Gene” refers to a nucleic acid fragment that expresses aspecific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence. “Native gene” refers to a gene as found in nature with its ownregulatory sequences “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure.

[0077] “Coding sequence” refers to a DNA sequence that codes for aspecific amino acid sequence. “Suitable regulatory sequences” refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters, translation leader sequences, introns, polyadenylationrecognition sequences, RNA processing site, effector binding site andstem-loop structure.

[0078] “Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters which cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

[0079] The “3′ non-coding sequences” refer to DNA sequences locateddownstream of a coding sequence and include polyadenylation recognitionsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor.

[0080] “RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be an RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA (mRNA)” refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to and derived from mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (U.S. Pat. No. 5,107,065;WO9928508). The complementarity of an antisense RNA may be with any partof the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, or the coding sequence. “Functional RNA” refers toantisense RNA, ribozyme RNA, or other RNA that is not translated yet hasan effect on cellular processes.

[0081] The term “operably linked” refers to the association of nucleicacid sequences on a single nucleic acid fragment so that the function ofone is affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., that the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

[0082] The term “expression”, as used herein, refers to thetranscription and stable accumulation of sense (mRNA) or antisense RNAderived from the nucleic acid fragment of the invention. Expression mayalso refer to translation of mRNA into a polypeptide.

[0083] “Transformation” refers to the transfer of a nucleic acidfragment into the genome of a host organism, resulting in geneticallystable inheritance. Host organisms containing the transformed nucleicacid fragments are referred to as “transgenic” or “recombinant” or“transformed” organisms.

[0084] The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes which are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA molecules. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

[0085] The term “altered biological activity” will refer to an activity,associated with a protein encoded by a microbial nucleotide sequencewhich can be measured by an assay method, where that activity is eithergreater than or less than the activity associated with the nativemicrobial sequence. “Enhanced biological activity” refers to an alteredactivity that is greater than that associated with the native sequence.“Diminished biological activity” is an altered activity that is lessthan that associated with the native sequence.

[0086] The term “sequence analysis software” refers to any computeralgorithm or software program that is useful for the analysis ofnucleotide or amino acid sequences. “Sequence analysis software” may becommercially available or independently developed. Typical sequenceanalysis software will include but is not limited to the GCG suite ofprograms (Wisconsin Package Version 9.0, Genetics Computer Group (GCG),Madison, Wis.), BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.215:403-410 (1990), and DNASTAR (DNASTAR, Inc. 1228 S. Park St. Madison,Wis. 53715 USA), and the FASTA program incorporating the Smith-Watermanalgorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int.Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor.Publisher: Plenum, New York, N.Y.). Within the context of thisapplication it will be understood that where sequence analysis softwareis used for analysis, that the results of the analysis will be based onthe “default values” of the program referenced, unless otherwisespecified. As used herein “default values” will mean any set of valuesor parameters which originally load with the software when firstinitialized.

[0087] Standard recombinant DNA and molecular cloning techniques usedhere are well known in the art and are described by Sambrook, J.,Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989) (hereinafter “Maniatis”); and by Silhavy, T. J., Bennan, M.L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring HarborLaboratory Cold Press Spring Harbor, N.Y. (1984); and by Ausubel, F. M.et al., Current Protocols in Molecular Biology, published by GreenePublishing Assoc. and Wiley-Interscience (1987).

[0088] The invention provides genes and gene products involved in thecarbon flux pathway of a Methylomonas sp. The invention alternativelyprovides methods of altering carbon flux in a methanotrophic bacteriacomprising the up-regulation or down-regulation of carbon flux either byintroducing the present genes into a host or by suppressing theexpression of sequence homologs to the present genes.

[0089] Isolation of Methylomonas 16a

[0090] The original environmental sample containing Methylomonas 16a wasobtained from pond sediment. The pond sediment was inoculated directlyinto a defined mineral medium under 25% methane in air. Methane was usedas the sole source of carbon and energy. Growth was followed until theoptical density at 660 nm was stable, whereupon the culture wastransferred to fresh medium such that a 1:100 dilution was achieved.After 3 successive transfers with methane as the sole carbon and energysource, the culture was plated onto defined minimal medium agar andincubated under 25% methane in air. Many methanotrophic bacterialspecies were isolated in this manner. However, Methylomonas 16a wasselected as the organism to study due to the rapid growth of colonies,large colony size, its ability to grow on minimal media, and pinkpigmentation indicative of an active biosynthetic pathway forcarotenoids.

[0091] Methanotrophs are classified into three metabolic groups (“TypeI”, “Type X” or “Type II”) based on the mode of carbon incorporation,morphology, % GC content and the presence or absence of key specificenzymes. Example 4, Table 2 shows key traits determined for Methylomonas16a in relation to the three major groupings of methanotrophs. Thestrain clearly falls into the Type I grouping based on every trait, withthe exception of nitrogen fixation. Available literature suggests thatthese organisms do not fix nitrogen. Therefore, Methylomonas 16a appearsto be unique in this aspect of nitrogen metabolism.

[0092] 16SrRNA extracted from the strain was sequenced and compared toknown 16SrRNAs from other microorganisms. The data showed 96% identityto sequences from Methylomonas sp. KSP III and Methylomonas sp. StrainLW13. Based on this evidence, as well as the other physiological traitsdescribed in Table 2, it was concluded that the strain was a member ofthe genus Methylomonas.

[0093] The present sequences have been identified by comparison ofrandom cDNA sequences to the GenBank database using the BLAST algorithmswell known to those skilled in the art. The nucleotide sequence of twogenes encoding fructose bisphosphate aldolase (FFBPA) have beenidentified. The gene sequences for these genes are given in SEQ ID NO:5and SEQ ID NO:7. The corresponding gene products are given in SEQ IDNO:6 and SEQ ID NO:8. Similarly, two genes encoding a transaldolaseassociated with the carbon flux pathway have been identified. Thesegenes are set forth in SEQ ID NO:1 and SEQ ID NO:3. Their correspondinggene products are set forth in SEQ ID NO:2 and SEQ ID NO:4. Additionallya gene encoding a keto deoxy phosphogluconate aldolase (KDPGA) has beenidentified and is given in SEQ ID NO:9 and the deduced amino acidsequence of the gene product is given in SEQ ID NO:10.

[0094] Genes encoding a phosphoglucomutase have also been identifiedwhere the genes and the corresponding gene products are given as SEQ IDNOs:11 and 12, respectively. Similarly, genes and gene products havebeen identified encoding a glucose 6 phosphate isomerase where the genesand their corresponding gene products are given as SEQ ID NO:13 and 14,respectively. Genes encoding a phosphofructokinase have also beenidentified where the genes and gene products are given as SEQ ID NOs:15and 16, respectively. A 6-phosphogluconate dehydratase encoding gene hasbeen identified and the gene and gene product are given in SEQ ID NOs:17and 18, respectively. Another carbon flux enzyme, 6-phosphogluconate 6phosphate I dehydrogenase, has been identified and the gene and geneproduct is given in SEQ ID NOs:19 and 20, respectively.

[0095] Accordingly, the present invention provides a Methylomonas sphaving a gene encoding a fructose bisphosphate aldolase (FBP aldolase),a keto deoxy phosphgogluconate/transaldolase (KDPG aldolase), aphosphoglucomutase, a glucose 6 phosphate isomerase, aphosphofructokinase, a 6-phosphogluconate dehydratase, and a6-phosphogluconate-6-phosphate 1 dehydrogenase.

[0096] More specifically the present strain is recognized as having agene encoding an transaldolase having about 78% identity at the aminoacid level over length of 328 amino acids using a Smith-Watermanalignment algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc.Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor.Publisher: Plenum, New York, N.Y.) to the sequence set forth in SEQ IDNO:2. More preferred amino acid fragments are at least about 80%-90%identical to the sequences herein. Most preferred are nucleic acidfragments that are at least 95% identical to the amino acid fragmentsreported herein. Similarly, preferred transaldolase encoding nucleicacid sequences corresponding to the instant seqeunces are those encodingactive proteins and which are at least 80% identical to the nucleic acidsequences of reported herein. More preferred transaldolase nucleic acidfragments are at least 90% identical to the sequences herein. Mostpreferred are transaldolase nucleic acid fragments that are at least 95%identical to the nucleic acid fragments reported herein.

[0097] More specifically the present strain is recognized as having agene encoding an transaldolase having about 50% identity at the aminoacid level over length of 160 amino acids using a Smith-Watermanalignment algorithm (W. R. Pearson, supra) to the sequence set forth inSEQ ID NO:4. More preferred amino acid fragments are at least about80%-90% identical to the sequences herein. Most preferred are nucleicacid fragments that are at least 95% identical to the amino acidfragments reported herein. Similarly, preferred transaldolase encodingnucleic acid sequences corresponding to the instant seqeunces are thoseencoding active proteins and which are at least 80% identical to thenucleic acid sequences of reported herein. More preferred transaldolasenucleic acid fragments are at least 90% identical to the sequencesherein. Most preferred are transaldolase nucleic acid fragments that areat least 95% identical to the nucleic acid fragments reported herein.

[0098] Additionally the present strain is recognized as having a geneencoding an FBP aldolase having about 76% identity at the amino acidlevel over length of 335 amino acids using a Smith-Waterman alignmentalgorithm (W. R. Pearson, supra) to the sequence set forth in SEQ IDNO:6. More preferred amino acid fragments are at least about 80%-90%identical to the sequences herein. Most preferred are nucleic acidfragments that are at least 95% identical to the amino acid fragmentsreported herein. Similarly, preferred FBP aldolase encoding nucleic acidsequences corresponding to the instant seqeunces are those encodingactive proteins and which are at least 80% identical to the nucleic acidsequences of reported herein. More preferred FBP aldolase nucleic acidfragments are at least 90% identical to the sequences herein. Mostpreferred are FBP aldolase nucleic acid fragments that are at least 95%identical to the nucleic acid fragments reported herein.

[0099] Additionally the present strain is recognized as having a geneencoding an FBP aldolase having about 40% identity at the amino acidlevel over length of 358 amino acids using a Smith-Waterman alignmentalgorithm (W. R. Pearson, supra) to the sequence set forth in SEQ IDNO:8. More preferred amino acid fragments are at least about 80%-90%identical to the sequences herein. Most preferred are nucleic acidfragments that are at least 95% identical to the amino acid fragmentsreported herein. Similarly, preferred FBP aldolase encoding nucleic acidsequences corresponding to the instant seqeunces are those encodingactive proteins and which are at least 80% identical to the nucleic acidsequences of reported herein. More preferred FBP aldolase nucleic acidfragments are at least 90% identical to the sequences herein. Mostpreferred are FBP aldolase nucleic acid fragments that are at least 95%identical to the nucleic acid fragments reported herein.

[0100] Additionally the present strain is recognized as having a geneencoding an KDPG aldolase having about 59% identity at the amino acidlevel over length of 212 amino acids using a Smith-Waterman alignmentalgorithm (W. R. Pearson, supra) to the sequence set forth in SEQ IDNO:10. More preferred amino acid fragments are at least about 80%-90%identical to the sequences herein. Most preferred are nucleic acidfragments that are at least 95% identical to the amino acid fragmentsreported herein. Similarly, preferred KDPG aldolase encoding nucleicacid sequences corresponding to the instant seqeunces are those encodingactive proteins and which are at least 80% identical to the nucleic acidsequences of reported herein. More preferred KDPG aldolase nucleic acidfragments are at least 90% identical to the sequences herein. Mostpreferred are KDPG aldolase nucleic acid fragments that are at least 95%identical to the nucleic acid fragments reported herein.

[0101] Additionally the present strain is recognized as having a geneencoding an phosphoglucomutase having about 65% identity at the aminoacid level over length of 545 amino acids using a Smith-Watermanalignment algorithm (W. R. Pearson, supra) to the sequence set forth inSEQ ID NO:12. More preferred amino acid fragments are at least about80%-90% identical to the sequences herein. Most preferred are nucleicacid fragments that are at least 95% identical to the amino acidfragments reported herein. Similarly, preferred phosphoglucomutaseencoding nucleic acid sequences corresponding to the instant seqeuncesare those encoding active proteins and which are at least 80% identicalto the nucleic acid sequences of reported herein. More preferredphosphoglucomutase nucleic acid fragments are at least 90% identical tothe sequences herein. Most preferred are phosphoglucomutase nucleic acidfragments that are at least 95% identical to the nucleic acid fragmentsreported herein.

[0102] Additionally the present strain is recognized as having a geneencoding an glucose-6-phosphate isomerase having about 64% identity atthe amino acid level over length of 592 amino acids using aSmith-Waterman alignment algorithm (W. R. Pearson, supra) to thesequence set forth in SEQ ID NO:14. More preferred amino acid fragmentsare at least about 80%-90% identical to the sequences herein. Mostpreferred are nucleic acid fragments that are at least 95% identical tothe amino acid fragments reported herein. Similarly, preferredglucose-6-phosphate isomerase encoding nucleic acid sequencescorresponding to the instant seqeunces are those encoding activeproteins and which are at least 80% identical to the nucleic acidsequences of reported herein. More preferred glucose-6-phosphateisomerase nucleic acid fragments are at least 90% identical to thesequences herein. Most preferred are glucose-6-phosphate isomerasenucleic acid fragments that are at least 95% identical to the nucleicacid fragments reported herein.

[0103] Additionally the present strain is recognized as having a geneencoding an phosphofructokinase having about 63% identity at the aminoacid level over length of 437 amino acids using a Smith-Watermanalignment algorithm (W. R. Pearson, supra) to the sequence set forth inSEQ ID NO:16. More preferred amino acid fragments are at least about80%-90% identical to the sequences herein. Most preferred are nucleicacid fragments that are at least 95% identical to the amino acidfragments reported herein. Similarly, preferred phosphofructokinaseencoding nucleic acid sequences corresponding to the instant seqeuncesare those encoding active proteins and which are at least 80% identicalto the nucleic acid sequences of reported herein. More preferredphosphofructokinase nucleic acid fragments are at least 90% identical tothe sequences herein. Most preferred are phosphofructokinase nucleicacid fragments that are at least 95% identical to the nucleic acidfragments reported herein.

[0104] Additionally the present strain is recognized as having a geneencoding an 6-phosphogluconate dehydratase having about 60% identity atthe amino acid level over length of 618 amino acids using aSmith-Waterman alignment algorithm (W. R. Pearson, supra) to thesequence set forth in SEQ ID NO:18. More preferred amino acid fragmentsare at least about 80%-90% identical to the sequences herein. Mostpreferred are nucleic acid fragments that are at least 95% identical tothe amino acid fragments reported herein. Similarly, preferred6-phosphogluconate dehydratase encoding nucleic acid sequencescorresponding to the instant seqeunces are those encoding activeproteins and which are at least 80% identical to the nucleic acidsequences of reported herein. More preferred 6-phosphogluconatedehydratase nucleic acid fragments are at least 90% identical to thesequences herein. Most preferred are 6-phosphogluconate dehydratasenucleic acid fragments that are at least 95% identical to the nucleicacid fragments reported herein.

[0105] Additionally the present strain is recognized as having a geneencoding an encoding a 6-phosphogluconate-6-phosphate-1-dehydrogenasehaving about 58% identity at the amino acid level over length of 501amino acids using a Smith-Waterman alignment algorithm (W. R. Pearson,supra) to the sequence set forth in SEQ ID NO:20. More preferred aminoacid fragments are at least about 80%-90% identical to the sequencesherein. Most preferred are nucleic acid fragments that are at least 95%identical to the amino acid fragments reported herein. Similarly,preferred 6-phosphogluconate-6-phosphate-1-dehydrogenase encodingnucleic acid sequences corresponding to the instant seqeunces are thoseencoding active proteins and which are at least 80% identical to thenucleic acid sequences of reported herein. More preferred6-phosphogluconate-6-phosphate-1-dehydrogenase nucleic acid fragmentsare at least 90% identical to the sequences herein. Most preferred are6-phosphogluconate-6-phosphate-1-dehydrogenase nucleic acid fragmentsthat are at least 95% identical to the nucleic acid fragments reportedherein.

[0106] Isolation of Homologs

[0107] The nucleic acid fragments of the instant invention may be usedto isolate genes encoding homologous proteins from the same or othermicrobial species. Isolation of homologous genes usingsequence-dependent protocols is well known in the art. Examples ofsequence-dependent protocols include, but are not limited to, methods ofnucleic acid hybridization, and methods of DNA and RNA amplification asexemplified by various uses of nucleic acid amplification technologies(e.g. polymerase chain reaction (PCR), Mullis et al., U.S. Pat.4,683,202), ligase chain reaction (LCR), Tabor, S. et al., Proc. Acad.Sci. USA 82, 1074, (1985)) or strand displacement amplification (SDA,Walker, et al., Proc. Natl. Acad. Sci. U.S.A., 89, 392, (1992)).

[0108] For example, genes encoding similar proteins or polypetides tothose of the instant invention could be isolated directly by using allor a portion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from any desired bacteria using methodologywell known to those skilled in the art. Specific oligonucleotide probesbased upon the instant nucleic acid sequences can be designed andsynthesized by methods known in the art (Maniatis). Moreover, the entiresequences can be used directly to synthesize DNA probes by methods knownto the skilled artisan such as random primers DNA labeling, nicktranslation, or end-labeling techniques, or RNA probes using availablein vitro transcription systems. In addition, specific primers can bedesigned and used to amplify a part of or full-length of the instantsequences. The resulting amplification products can be labeled directlyduring amplification reactions or labeled after amplification reactions,and used as probes to isolate full length DNA fragments under conditionsof appropriate stringency.

[0109] Typically, in PCR-type amplification techniques, the primers havedifferent sequences and are not complementary to each other. Dependingon the desired test conditions, the sequences of the primers should bedesigned to provide for both efficient and faithful replication of thetarget nucleic acid. Methods of PCR primer design are common and wellknown in the art (Thein and Wallace, “The use of oligonucleotide asspecific hybridization probes in the Diagnosis of Genetic Disorders”, inHuman Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986)pp. 33-50 IRL Press, Herndon, Va.; Rychlik, W. (1993) In White, B. A.(ed.), Methods in Molecular Biology, Vol. 15, pages 31-39, PCRProtocols: Current Methods and Applications. Humania Press, Inc.,Totowa, N.J.).

[0110] Generally two short segments of the instant sequences may be usedin polymerase chain reaction protocols to amplify longer nucleic acidfragments encoding homologous genes from DNA or RNA. The polymerasechain reaction may also be performed on a library of cloned nucleic acidfragments wherein the sequence of one primer is derived from the instantnucleic acid fragments, and the sequence of the other primer takesadvantage of the presence of the polyadenylic acid tracts to the 3′ endof the mRNA precursor encoding microbial genes. Alternatively, thesecond primer sequence may be based upon sequences derived from thecloning vector. For example, the skilled artisan can follow the RACEprotocol (Frohman et al., PNAS USA 85:8998 (1988)) to generate cDNAs byusing PCR to amplify copies of the region between a single point in thetranscript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′directions can be designed from the instant sequences. Usingcommercially available 3′ RACE or 5′ RACE systems (BRL), specific 3′ or5′ cDNA fragments can be isolated (Ohara et al., PNAS USA 86:5673(1989); Loh et al., Science 243:217 (1989)).

[0111] Alternatively the instant sequences may be employed ashybridization reagents for the identification of homologs. The basiccomponents of a nucleic acid hybridization test include a probe, asample suspected of containing the gene or gene fragment of interest,and a specific hybridization method. Probes of the present invention aretypically single stranded nucleic acid sequences which are complementaryto the nucleic acid sequences to be detected. Probes are “hybridizable”to the nucleic acid sequence to be detected. The probe length can varyfrom 5 bases to tens of thousands of bases, and will depend upon thespecific test to be done. Typically a probe length of about 15 bases toabout 30 bases is suitable. Only part of the probe molecule need becomplementary to the nucleic acid sequence to be detected. In addition,the complementarity between the probe and the target sequence need notbe perfect. Hybridization does occur between imperfectly complementarymolecules with the result that a certain fraction of the bases in thehybridized region are not paired with the proper complementary base.

[0112] Hybridization methods are well defined. Typically the probe andsample must be mixed under conditions which will permit nucleic acidhybridization. This involves contacting the probe and sample in thepresence of an inorganic or organic salt under the proper concentrationand temperature conditions. The probe and sample nucleic acids must bein contact for a long enough time that any possible hybridizationbetween the probe and sample nucleic acid may occur. The concentrationof probe or target in the mixture will determine the time necessary forhybridization to occur. The higher the probe or target concentration theshorter the hybridization incubation time needed. Optionally achaotropic agent may be added. The chaotropic agent stabilizes nucleicacids by inhibiting nuclease activity. Furthermore, the chaotropic agentallows sensitive and stringent hybridization of short oligonucleotideprobes at room temperature [Van Ness and Chen (1991) Nucl. Acids Res.19:5143-5151]. Suitable chaotropic agents include guanidinium chloride,guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate,sodium perchlorate, rubidium tetrachloroacetate, potassium iodide, andcesium trifluoroacetate, among others. Typically, the chaotropic agentwill be present at a final concentration of about 3M. If desired, onecan add formamide to the hybridization mixture, typically 30-50% (v/v).

[0113] Various hybridization solutions can be employed. Typically, thesecomprise from about 20 to 60% volume, preferably 30%, of a polar organicsolvent. A common hybridization solution employs about 30-50% v/vformamide, about 0.15 to 1M sodium chloride, about 0.05 to 0.1M buffers,such as sodium citrate, Tris-HCl, PIPES or HEPES (pH range about 6-9),about 0.05 to 0.2% detergent, such as sodium dodecylsulfate, or between0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kilodaltons),polyvinylpyrrolidone (about 250-500 kdal), and serum albumin. Alsoincluded in the typical hybridization solution will be unlabeled carriernucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA, e.g.,calf thymus or salmon sperm DNA, or yeast RNA, and optionally from about0.5 to 2% wt./vol. glycine. Other additives may also be included, suchas volume exclusion agents which include a variety of polarwater-soluble or swellable agents, such as polyethylene glycol, anionicpolymers such as polyacrylate or polymethylacrylate, and anionicsaccharidic polymers, such as dextran sulfate.

[0114] Nucleic acid hybridization is adaptable to a variety of assayformats. One of the most suitable is the sandwich assay format. Thesandwich assay is particularly adaptable to hybridization undernon-denaturing conditions. A primary component of a sandwich-type assayis a solid support. The solid support has adsorbed to it or covalentlycoupled to it immobilized nucleic acid probe that is unlabeled andcomplementary to one portion of the sequence.

[0115] Recombinant Expression—Microbial

[0116] The genes and gene products of the instant sequences may beproduced in heterologous host cells, particularly in the cells ofmicrobial hosts. Expression in recombinant microbial hosts may be usefulfor the expression of various pathway intermediates, for the modulationof pathways already existing in the host, and for the synthesis of newproducts heretofore not possible using the host. Additionally, the geneproducts may be useful for conferring higher growth yields of the hostor for enabling alternative growth modes to be utilized.

[0117] Preferred heterologous host cells for expression of the instantgenes and nucleic acid molecules are microbial hosts that can be foundbroadly within microbial families and which grow over a wide range oftemperatures, pH values, and solvent tolerances. Such microbes willinclude generally bacteria, yeast, and filamentous fungi. Specifically,suitable yeasts and fungi will include, but are not limited to,Aspergillus, Saccharomyces, Pichia, Candida, and Hansenula. Suitablebacterial species include, but are not limited to, Salmonella, Bacillus,Acinetobacter, Rhodococcus, Streptomyces, Escherichia, and Pseudomonas.Most preferred hosts for the expression of the present carbon flux genesare members of the methanotrophic class of bacteria includingMethylomonas, Methylococcus and Methylobacter. Particularly suited fortransformation will be members of the genus Methylomonas. Thesebacterial species have the ability to convert single carbon substratessuch as methane and methanol to useful products and these genes areparticularly suited for substrates found in these hosts.

[0118] Of particular interest in the present invention are high growthobligate methanotrophs having an energetically favorable carbon fluxpathway. For example, Applicants have discovered a specific strain ofmethanotroph having several pathway features which make it particularlyuseful for carbon flux manipulation. This type of strain has served asthe host in the present application and is known as Methylomonas 16a(ATCC PTA 2402).

[0119] The present strain contains several anomalies in the carbonutilization pathway. For example, based on genome sequence data, thestrain is shown to contain genes for two pathways of hexose metabolism.The Entner-Douderoff pathway which utilizes the keto-deoxyphosphogluconate aldolase enzyme is present in the strain. It isgenerally well accepted that this is the operative pathway in obligatemethanotrophs. Also present, however, is the Embden-Meyerhof pathwaywhich utilizes the fructose bisphosphate aldolase enzyme. It is wellknown that this pathway is either not present or not operative inobligate methanotrophs. Energetically, the latter pathway is mostfavorable and allows greater yield of biologically useful energy andultimately production of cell mass and other cell mass-dependentproducts in Methylomonas 16a. The activity of this pathway in thepresent 16a strain has been confirmed through microarray data andbiochemical evidence measuring the reduction of ATP. Although the 16astrain has been shown to possess both the Embden-Meyerhof and theEntner-Douderoff pathway enzymes, the data suggests that theEmbden-Meyerhof pathway enzymes are more strongly expressed than theEntner-Douderoff pathway enzymes. This result is surprising and counterto existing beliefs concerning the glycolytic metabolism ofmethanotrophic bacteria. Applicants have discovered other methanotrophicbacteria having this characteristic, including for example, Methylomonasclara and Methylosinus sporium. It is likely that this activity hasremained undiscovered in methanotrophs due to the lack of activity ofthe enzyme with ATP, the typical phosphoryl donor for the enzyme in mostbacterial systems.

[0120] A particularly novel and useful feature of the Embden-Meyerhofpathway in strain 16a is that the key phosphofructokinase step ispyrophosphate dependent instead of ATP dependent. This feature adds tothe energy yield of the pathway by using pyrophosphate instead of ATP.Because of its significance in providing an energetic advantage to thestrain this gene in the carbon flux pathway is considered diagnostic forthe present strain.

[0121] Comparison of the pyrophosphate dependent phosphofructokinasegene sequence (SEQ ID No:15) and deduced amino acid sequence (SEQ IDNO:16) to public databases reveals that the most similar known sequencesare about 63% identical to the amino acid sequence reported herein overa length of 437 amino acids using a Smith-Waterman alignment algorithm(W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994),Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum,New York, N.Y.). More preferred amino acid fragments are at least about80%-90% identical to the sequences herein. Most preferred are nucleicacid fragments that are at least 95% identical to the amino acidfragments reported herein. Similarly, preferred pyrophosphate dependentphosphofructokinase encoding nucleic acid sequences corresponding to theinstant gene are those encoding active proteins and which are at least80% identical to the nucleic acid sequences of reported herein. Morepreferred pyrophosphate dependent phosphofructokinase nucleic acidfragments are at least 90% identical to the sequences herein. Mostpreferred are pyrophosphate dependent phosphofructokinase nucleic acidfragments that are at least 95% identical to the nucleic acid fragmentsreported herein.

[0122] In methanotrophic bacteria methane is converted to biomoleculesvia a cyclic set of reactions known as the ribulose monophosphatepathway or RuMP cycle. This pathway is comprised of three phases, eachphase being a series of enzymatic steps (FIG. 1). The first step is“fixation” or incorporation of C-1 (formaldehyde) into a pentose to forma hexose or six-carbon sugar. This occurs via a condensation reactionbetween a 5-carbon sugar (pentose) and formaldehyde and is catalyzed byhexulose monophosphate synthase. The second phase is termed “cleavage”and results in splitting of that hexose into two 3-carbon molecules. Oneof those three-carbon molecules is recycled back through the RuMPpathway and the other 3-carbon fragment is utilized for cell growth. Inmethanotrophs and methylotrophs the RuMP pathway may occur as one ofthree variants. However, only two of these variants are commonly found:the FBP/TA (fructose bisphosphotase/Transaldolase) or the KDPG/TA (ketodeoxy phosphogluconate/transaldolase) pathway (Dijkhuizen L., G. E.Devries. The Physiology and biochemistry of aerobic methanol-utilizinggram negative and gram positive bacteria. In: Methane and MethanolUtilizers 1992, ed Colin Murrell and Howard Dalton Plenum Press NY).

[0123] The present strain is unique in the way it handles the “cleavage”steps where genes were found that carry out this conversion via fructosebisphosphate as a key intermediate. The genes for fructose bisphosphatealdolase and transaldolase were found clustered together on one piece ofDNA. Secondly, the genes for the other variant involving the keto deoxyphosphogluconate intermediate were also found clustered together.Available literature teaches that these organisms (obligatemethylotrophs and methanotrophs) rely solely on the KDPG pathway andthat the FBP-dependent fixation pathway is utilized by facultativemethylotrophs (Dijkhuizen et al., supra). Therefore the latterobservation is expected whereas the former is not. The finding of theFBP genes in an obligate methane utilizing bacterium is both surprisingand suggestive of utility. The FBP pathway is energetically favorable tothe host microorganism due to the fact that more energy (ATP) isutilized than is utilized in the KDPG pathway. Thus organisms thatutilize the FBP pathway may have an energetic advantage and growthadvantage over those that utilize the KDPG pathway. This advantage mayalso be useful for energy-requiring production pathways in the strain.By using this pathway, a methane-utilizing bacterium may have anadvantage over other methane utilizing organisms as production platformsfor either single cell protein or for any other product derived from theflow of carbon through the RuMP pathway.

[0124] Accordingly the present invention provides a method for alteringcarbon flux in a high growth, energetically favorable Methylomonasstrain which

[0125] (a) grows on a C1 carbon substrate selected from the groupconsisting of methane and methanol; and

[0126] (b) comprises a functional Embden-Meyerhof carbon pathway, saidpathway comprising a gene encoding a pyrophosphate dependentphosphofructokinase enzyme.

[0127] Microbial expression systems and expression vectors containingregulatory sequences that direct high level expression of foreign genesare well known to those skilled in the art. Any of these could be usedto construct chimeric genes for expression of the any of the presentgenes. These chimeric genes could then be introduced into appropriatemicroorganisms via transformation to provide recombinant expression ofthe enzymes and manipulation of the carbon pathways.

[0128] Vectors or cassettes useful for the transformation of suitablehost cells are well known in the art. Typically the vector or cassettecontains sequences directing transcription and translation of therelevant gene, a selectable marker, and sequences allowing autonomousreplication or chromosomal integration. Suitable vectors comprise aregion 5′ of the gene which harbors transcriptional initiation controlsand a region 3′ of the DNA fragment which controls transcriptionaltermination. It is most preferred when both control regions are derivedfrom genes homologous to the transformed host cell, although it is to beunderstood that such control regions need not be derived from the genesnative to the specific species chosen as a production host.

[0129] Initiation control regions or promoters, which are useful todrive expression of the instant ORF's in the desired host cell arenumerous and familiar to those skilled in the art. Virtually anypromoter capable of driving these genes is suitable for the presentinvention including but not limited to CYC1, HIS3, GAL1, GAL10, ADH1,PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful forexpression in Saccharomyces); AOX1 (useful for expression in Pichia);and lac, ara, tet, trp, IP_(L), IP_(R), T7, tac, and trc (useful forexpression in Escherichia coli) as well as the amy, apr, npr promotersand various phage promoters useful for expression in Bacillus.

[0130] Termination control regions may also be derived from variousgenes native to the preferred hosts. Optionally, a termination site maybe unnecessary, however, it is most preferred if included.

[0131] Pathway Engineering

[0132] The present genes may be used to affect carbon flow in bacteriaand specifically methanotrophic bacteria. Commercial applications of themethanotrops have revolved around the production of single cell protein(Villadsen, John, Recent Trends Chem. React. Eng., [Proc. Int. Chem.React. Eng. Conf.], 2nd (1987), Volume 2, 320-33. Editor(s): Kulkarni,B. D.; Mashelkar, R. A.; Sharma, M. M. Publisher: Wiley East, New Delhi,India; Naguib, M.,Proc. OAPEC Symp. Petroprotein, [Pap.] (1980), MeetingDate 1979, 253-77, Publisher: Organ. Arab Pet. Exporting Countries,Kuwait, Kuwait) and the epoxidation of alkenes for production ofchemicals (U.S. Pat. No. 4,348,476). These C1 substrate utilizingbacteria also are known to produce polysaccharides, used as thickenersin food and non-food industries, and isoprenoid compounds and carotenoidpigments of various carbon lengths (Urakami et al., J. Gen. Appl.Microbiol. (1986), 32(4), 317-41). The production of all of thesecommercially useful products will be impacted by alterations in carbonflux, in general, and by manipulation of the present genes, inparticular. Such manipulation may be effected by the up- ordown-regulation of various members of the carbon flux pathway.

[0133] Many of the key genes in the carbon utilization pathway are nowdisclosed in the present invention. Referring to FIG. 1, for example,the present invention provides genes encoding two distinct carbon fluxpathways isolated from a methanotrophic bacteria. The genes and geneproducts are set forth in SEQ ID NO:1-SEQ ID NO:20, and encode both aKDPG aldolase and a FBP aldolase as well as a phosphoglucomutase,pyrophosphate dependent phosphofructokinase pyrophosphate,6-phosphogluconate dehydratase, and a glucose 6 phosphate 1dehydrogenase. The phosphoglucomutase is responsible for theinterconversion of glucose-6-phosphate to glucose-1-phosphate, whichfeeds into either the Entner douderoff or Embden-Meyerhof carbon fluxpathways. As shown in FIG. 1, fructose-6-phosphate may be converted toeither glucose-6-phosphase by glucose phophate isomerase(Entner-Douderoff) or to fructose-1,6-bisphosphate (FBP) by aphosphofructokinase (Embden-Meyerhof). Following the Embden-Meyerhofpathway, FBP is then taken to two three-carbon moieties,dihydroxyacetone and 3-phosphoglyceraldehyde by the FBP aldolase.Returning to the Entner-Douderoff pathway, glucose-6-phosphate is takento 6-phosphogluconate by a glucose-6-phosphate dehydrogenase which issubsequently taken to 2-keto-3-deoxy-6-phosphogluconate (KDPG) by a 6phosphogluconate dehydratase. The KDPG is then converted to twothree-carbon moieties (pyruvate and 3-phosphoglyceraldehyde) by a KDPGaldolase. Thus, the Embden-Meyerhof and Entner-Douderoff pathways arerejoined at the level of 3-phosphoglyceraldehyde. Manipulations in anyone or all of these genes may be used for commercial advantage in theproduction of materials from a variety of bacteria and most suitablyfrom methanotrophic bacteria.

[0134] Methods of manipulating genetic pathways are common and wellknown in the art. Selected genes in a particularly pathway may beupregulated or down regulated by variety of methods. Additionally,competing pathways in the organism may be eliminated or sublimated bygene disruption and similar techniques.

[0135] Once a key genetic pathway has been identified and sequenced,specific genes may be upregulated to increase the output of the pathway.For example, additionally copies of the targeted genes may be introducedinto the host cell on multicopy plasmids such as pBR322. Alternativelythe target genes may be modified so as to be under the control ofnon-native promoters. Where it is desired that a pathway operate at aparticular point in a cell cycle or during a fermentation run, regulatedor inducible promoters may used to replace the native promoter of thetarget gene. Similarly, in some cases the native or endogenous promotermay be modified to increase gene expression. For example, endogenouspromoters can be altered in vivo by mutation, deletion, and/orsubstitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al.,PCT/US93/03868).

[0136] Alternatively it may be necessary to reduce or eliminate theexpression of certain genes in the target pathway or in competingpathways that may serve as competing sinks for energy or carbon. Methodsof down-regulating genes for this purpose have been explored. Wheresequence of the gene to be disrupted is known, one of the most effectivemethods gene down regulation is targeted gene disruption where foreignDNA is inserted into a structural gene so as to disrupt transcription.This can be effected by the creation of genetic cassettes comprising theDNA to be inserted (often a genetic marker) flanked by sequence having ahigh degree of homology to a portion of the gene to be disrupted.Introduction of the cassette into the host cell results in insertion ofthe foreign DNA into the structural gene via the native DNA replicationmechanisms of the cell. (See for example Hamilton et al. (1989) J.Bacteriol. 171:4617-4622, Balbas et al. (1993) Gene 136:211-213,Gueldener et al. (1996) Nucleic Acids Res. 24:2519-2524, and Smith etal. (1996) Methods Mol. Cell. Biol. 5:270-277.)

[0137] Antisense technology is another method of down regulating geneswhere the sequence of the target gene is known. To accomplish this, anucleic acid segment from the desired gene is cloned and operably linkedto a promoter such that the anti-sense strand of RNA will betranscribed. This construct is then introduced into the host cell andthe antisense strand of RNA is produced. Antisense RNA inhibits geneexpression by preventing the accumulation of mRNA which encodes theprotein of interest. The person skilled in the art will know thatspecial considerations are associated with the use of antisensetechnologies in order to reduce expression of particular genes. Forexample, the proper level of expression of antisense genes may requirethe use of different chimeric genes utilizing different regulatoryelements known to the skilled artisan.

[0138] Although targeted gene disruption and antisense technology offereffective means of down regulating genes where the sequence is known,other less specific methodologies have been developed that are notsequence based. For example, cells may be exposed to a UV radiation andthen screened for the desired phenotype. Mutagenesis with chemicalagents is also effective for generating mutants and commonly usedsubstances include chemicals that affect nonreplicating DNA such as HNO₂and NH₂OH, as well as agents that affect replicating DNA such asacridine dyes, notable for causing frameshift mutations. Specificmethods for creating mutants using radiation or chemical agents are welldocumented in the art. See for example Thomas D. Brock in Biotechnology:A Textbook of Industrial Microbiology, Second Edition (1989) SinauerAssociates, Inc., Sunderland, Mass., or Deshpande, Mukund V., Appl.Biochem. Biotechnol., 36, 227, (1992).

[0139] Another non-specific method of gene disruption is the use oftransposoable elements or transposons. Transposons are genetic elementsthat insert randomly in DNA but can be latter retrieved on the basis ofsequence to determine where the insertion has occurred. Both in vivo andin vitro transposition methods are known. Both methods involve the useof a transposable element in combination with a transposase enzyme. Whenthe transposable element or transposon, is contacted with a nucleic acidfragment in the presence of the transposase, the transposable elementwill randomly insert into the nucleic acid fragment. The technique isuseful for random mutageneis and for gene isolation, since the disruptedgene may be identified on the basis of the sequence of the transposableelement. Kits for in vitro transposition are commercially available (seefor example The Primer Island Transposition Kit, available from PerkinElmer Applied Biosystems, Branchburg, N.J., based upon the yeast Ty1element; The Genome Priming System, available from New England Biolabs,Beverly, Mass.; based upon the bacterial transposon Tn7; and the EZ::TNTransposon Insertion Systems, available from Epicentre Technologies,Madison, Wis., based upon the Tn5 bacterial transposable element.

[0140] Within the context of the present invention it may be useful tomodulate the expression of the carbon flux pathway. It is apparent fromthe known pathways in methanotrophic bacteria that there can be utilityin either the FBP/TA or KDGP/TA pathway, depending on the targetproduct. The FBP/TA pathway is more energy-yielding and thus isadvantageous from the standpoint of producing more cellular mass perunit of methane metabolized. Thus if the strain is forced to utilizethis pathway via a gene knock-out of the KDGP/TA pathway, it isanticipated that greater cell mass will be produced. In addition, theproduction of chemicals that have a high energy requirement forbiosynthesis in the form of ATP may also be enhanced by deletion ormutation of the KDGP/TA pathway. Chemical production requiring pyruvateas a key intermediate, however, might benefit from the deletion orknock-out of the FBP/TA pathway genes. As an integral part of theMethylomonas production platform it is desirable to have the capabilityto utilize either pathway via introduction of specialized regulatorygene promoters that will enable either pathway to be switched on or offin the presence of chemicals that could be added to the fermentation.

[0141] More specifically, it has been noted that the presentMethylomonas 16a comprises genes encoding both the Entner-Douderoff andEmbden-Meyerhof carbon flux pathways. Because the Embden-Meyerhofpathway is more energy efficient it may be desirable to over-express thegenes in this pathway. Additionally, it is likely that theEntner-Douderoff pathway is a competitive pathway and inhibition of thispathway may lead to increased energy efficiency in the Embden-Meyerhofsystem. This might be accomplished by selectively using the abovedescribed methods of gene down regulation on the sequence encoding theketo-deoxy phosphogluconate aldolase (SEQ ID No:9) or any of the othermembers of the Entner-Ddouderoff system and upregulating the geneencoding the fructose bisphosphatase aldolase of the Embden-Meyerhofsystem (SEQ ID NO:5 OR 7). In this fashion, the carbon flux in thepresent Methylomonas 16a may be optimized. Additionally, where thepresent strain has been engineered to produce specific organic materialssuch as aromatics for monomer production, optimization of the carbonflux pathway will lead to increased yields of these materials.

[0142] Industrial Scale Production

[0143] Where the engineering of a commercial bacterial productionplatform comprising the present genes is desired, a variety of culturemethodologies may be applied. For example, large scale production of aspecific product or products from a recombinant microbial host may beproduced by both batch or continuous culture methodologies

[0144] A classical batch culturing method is a closed system where thecomposition of the media is set at the beginning of the culture and notsubject to artificial alterations during the culturing process. Thus, atthe beginning of the culturing process the media is inoculated with thedesired organism or organisms and growth or metabolic activity ispermitted to occur adding nothing to the system. Typically, however, a“batch” culture is batch with respect to the addition of carbon sourceand attempts are often made at controlling factors such as pH and oxygenconcentration. In batch systems the metabolite and biomass compositionsof the system change constantly up to the time the culture isterminated. Within batch cultures cells moderate through a static lagphase to a high growth log phase and finally to a stationary phase wheregrowth rate is diminished or halted. If untreated, cells in thestationary phase will eventually die. Cells in log phase are oftenresponsible for the bulk of production of end product or intermediate insome systems. Stationary or post-exponential phase production can beobtained in other systems.

[0145] A variation on the standard batch system is the Fed-Batch system.Fed-Batch culture processes are also suitable in the present inventionand comprise a typical batch system with the exception that thesubstrate is added in increments as the culture progresses. Fed-Batchsystems are useful when catabolite repression is apt to inhibit themetabolism of the cells and where it is desirable to have limitedamounts of substrate in the media. Measurement of the actual substrateconcentration in Fed-Batch systems is difficult and is thereforeestimated on the basis of the changes of measurable factors such as pH,dissolved oxygen and the partial pressure of waste gases such as CO₂.Batch and Fed-Batch culturing methods are common and well known in theart and examples may be found in Thomas D. Brock in Biotechnology: ATextbook of Industrial Microbiology, Second Edition (1989) SinauerAssociates, Inc., Sunderland, Mass., or Deshpande, Mukund V., Appl.Biochem. Biotechnol., 36, 227, (1992), herein incorporated by reference.

[0146] Commercial use of the instant gene pathways may also beaccomplished with a continuous culture. Continuous cultures are an opensystem where a defined culture media is added continuously to abioreactor and an equal amount of conditioned media is removedsimultaneously for processing. Continuous cultures generally maintainthe cells at a constant high liquid phase density where cells areprimarily in log phase growth. Alternatively continuous culture may bepracticed with immobilized cells where carbon and nutrients arecontinuously added and valuable products, by-products or waste productscontinuously removed from the cell mass. Cell immobilization may beperformed using a wide range of solid supports composed of naturaland/or synthetic materials.

[0147] Continuous or semi-continuous culture allows for the modulationof one factor or any number of factors that affect cell growth or endproduct concentration. For example, one method will maintain a limitingnutrient such as the carbon source or nitrogen level at a fixed rate andallow all other parameters to moderate. In other systems a number offactors affecting growth can be altered continuously while the cellconcentration, measured by media turbidity, is kept constant. Continuoussystems strive to maintain steady state growth conditions and thus thecell loss due to media being drawn off must be balanced against the cellgrowth rate in the culture. Methods of modulating nutrients and growthfactors for continuous culture processes as well as techniques formaximizing the rate of product formation are well known in the art ofindustrial microbiology and a variety of methods are detailed by Brock,supra.

[0148] Protein Engineering

[0149] It is contemplated that the present nucleotide sequences may beused to produce gene products having enhanced or altered activity.Various methods are known for mutating a native gene sequence to producea gene product with altered or enhanced activity including but notlimited to error prone PCR (Melnikov et al., Nucleic Acids Research,(Feb. 15, 1999) Vol. 27, No. 4, pp. 1056-1062); site directedmutagenesis (Coombs et al., Proteins (1998), 259-311, 1 plate.Editor(s): Angeletti, Ruth Hogue. Publisher: Academic, San Diego, Calif.and “gene shuffling” (U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721;and 5,837,458, incorporated herein by reference).

[0150] The method of gene shuffling is particularly attractive due toits facile implementation, and high rate of mutagenesis and ease ofscreening. The process of gene shuffling involves the restrictionendonuclease cleavage of a gene of interest into fragments of specificsize in the presence of additional populations of DNA regions of bothsimilarity to or difference to the gene of interest. This pool offragments will then be denatured and reannealed to create a mutatedgene. The mutated gene is then screened for altered activity.

[0151] The carbon flux sequences of the present invention may be mutatedand screened for altered or enhanced activity by this method. Thesequences should be double stranded and can be of various lengthsranging form 50 bp to 10 kb. The sequences may be randomly digested intofragments ranging from about 10 bp to 1000 bp, using restrictionendonucleases well known in the art (Maniatis supra). In addition to theinstant microbial sequences, populations of fragments that arehybridizable to all or portions of the microbial sequence may be added.Similarly, a population of fragments which are not hybridizable to theinstant sequence may also be added. Typically these additional fragmentpopulations are added in about a 10 to 20 fold excess by weight ascompared to the total nucleic acid. Generally if this process isfollowed the number of different specific nucleic acid fragments in themixture will be about 100 to about 1000. The mixed population of randomnucleic acid fragments are denatured to form single-stranded nucleicacid fragments and then reannealed. Only those single-stranded nucleicacid fragments having regions of homology with other single-strandednucleic acid fragments will reanneal. The random nucleic acid fragmentsmay be denatured by heating. One skilled in the art could determine theconditions necessary to completely denature the double stranded nucleicacid. Preferably the temperature is from 80° C. to 100° C. The nucleicacid fragments may be reannealed by cooling. Preferably the temperatureis from 20° C. to 75° C. Renaturation can be accelerated by the additionof polyethylene glycol (“PEG”) or salt. A suitable salt concentrationmay range from 0 mM to 200 mM. The annealed nucleic acid fragments arenext incubated in the presence of a nucleic acid polymerase and dNTP's(i.e. dATP, dCTP, dGTP and dTTP). The nucleic acid polymerase may be theKlenow fragment, the Taq polymerase or any other DNA polymerase known inthe art. The polymerase may be added to the random nucleic acidfragments prior to annealing, simultaneously with annealing or afterannealing. The cycle of denaturation, renaturation and incubation in thepresence of polymerase is repeated for a desired number of times.Preferably the cycle is repeated from 2 to 50 times, more preferably thesequence is repeated from 10 to 40 times. The resulting nucleic acid isa larger double-stranded polynucleotide of from about 50 bp to about 100kb and may be screened for expression and altered activity by standardcloning and expression protocol. (Maniatis supra).

[0152] Gene Expression Profiling

[0153] The present carbon flux genes may be used in connection with geneexpression profiling technology for metabolic characterization of thecell from which the genes came. For example, many external changes suchas changes in growth condition or exposure to chemicals can causeinduction or repression of genes in the cell. The induction orrepression of genes can be used for a screening system to determine thebest growth conditions for a production organism and drug discovery withsimilar mode of action compound, just to mention a few. On the otherhand, by amplifying or disrupting genes, one can manipulate theproduction of the amount of cellular products as well as the timelineupon which those products are produced. All or a portion of the presentnucleic acid fragments of the instant invention may be used as probesfor gene expression monitoring and gene expression profiling.

[0154] For example, all or a portion of the instant nucleic acidfragments may be immobilized on a nylon membrane or a glass slide. AGeneration II DNA spotter (Molecular Dynamics) is one of the availabletechnologies to array the DNA samples onto the coated glass slides.Other array methods are also available and well known in the art. Afterthe cells are grown in various growth conditions or treated withpotential candidates, cellular RNA is purified. Fluorescent orradioactive labeled target cDNA can be made by reverse transcription ofmRNA. The target mixture is hybridized to the probes and washed usingconditions well known in the art. The amount of the target geneexpression is quantified by the intensity of radioactivity orfluorescence labels (e.g., confocal laser microscope: MolecularDynamics). The intensities of radioactivity or fluorescent label at theimmobilized probes are measured using technology well known in the art.The two color fluorescence detection scheme (e.g., Cy3 and Cy5) has theadvantage over radioactively labeled targets by allowing rapid andsimultaneous differential expression analysis of independent samples. Inaddition, the use of ratio measurements compensates for probe to probevariation of intensity due to DNA concentration and hybridizationefficiency. In the case of fluorescence labeling, the two fluorescentimages obtained with the appropriate excitation and emission filtersconstitute the raw data from which differential gene expression ratiovalues are calculated. The intensity of images are analyzed using theavailable software (e.g., Array Vision 4.0: Imaging Research Inc.) wellknown in the art and normalized to compensate for the differentialefficiencies of labeling and detection of the label. There are manydifferent ways known in the art to normalize the signals. One of theways to normalize the signal is by correcting the signal againstinternal controls. Another way is to run a separate array with labeledgenomic driven DNA and compare the signal with mRNA driven signals. Thismethod also allows measurement of the transcript abundance. The arraydata of individual genes is examined and evaluated to determine theinduction or repression of each gene under the test conditions.

EXAMPLES

[0155] The present invention is further defined in the followingExamples. It should be understood that these Examples, while indicatingpreferred embodiments of the invention, are given by way of illustrationonly. From the above discussion and these Examples, one skilled in theart can ascertain the essential characteristics of this invention, andwithout departing from the spirit and scope thereof, can make variouschanges and modifications of the invention to adapt it to various usagesand conditions.

[0156] General Methods

[0157] Standard recombinant DNA and molecular cloning techniques used inthe Examples are well known in the art and are described by Sambrook,J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A LaboratoryManual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989)(Maniatis) and by T. J. Silhavy, M. L. Bennan, and L. W. Enquist,Experiments with Gene Fusions, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1984) and by Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, pub. by Greene Publishing Assoc. andWiley-Interscience (1987).

[0158] Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Techniques suitable foruse in the following examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, eds), American Society for Microbiology, Washington,D.C. (1994)) or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, Second Edition, Sinauer Associates, Inc.,Sunderland, Mass. (1989). All reagents, restriction enzymes andmaterials used for the growth and maintenance of bacterial cells wereobtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO Laboratories(Detroit, Mich.), GIBCO/BRL (Gaithersburg, Md.), or Sigma ChemicalCompany (St. Louis, Mo.) unless otherwise specified.

[0159] Manipulations of genetic sequences were accomplished using thesuite of programs available from the Genetics Computer Group Inc.(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.). Where the GCG program “Pileup” was used the gap creation defaultvalue of 12, and the gap extension default value of 4 were used. Wherethe CGC “Gap” or “Bestfit” programs were used the default gap creationpenalty of 50 and the default gap extension penalty of 3 were used. Inany cases where GCG program parameters were not prompted for, in theseor any other GCG program, default values were used.

[0160] Multiple alignment of the sequences was performed using the FASTAprogram incorporating the Smith-Waterman algorithm (W. R. Pearson,Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York,N.Y.).

[0161] The meaning of abbreviations is as follows: “h” means hour(s),“min” means minute(s), “sec” means second(s), “d” means day(s), “mL”means milliliters, “L” means liters.

Example 1 Isolation Of Methylomonas 16a

[0162] The original environmental sample containing the isolate wasobtained from pond sediment. The pond sediment was inoculated directlyinto a defined mineral medium under 25% methane in air. Methane was thesole source of carbon and energy. Growth was followed until the opticaldensity at 660 nm was stable whereupon the culture was transferred tofresh medium such that a 1:100 dilution was achieved. After 3 successivetransfers with methane as sole carbon and energy source the culture wasplated onto defined minimal medium agar and incubated under 25% methanein air. Many methanotrophic bacterial species were isolated in thismanner. However, Methylomonas 16a was selected as the organism to studydue to the rapid growth of colonies, large colony size, ability to growon minimal media, and pink pigmentation indicative of an activebiosynthetic pathway for carotenoids.

Example 2 Preparation of Genomic DNA for Sequencing and SeqeunceGeneration

[0163] Genomic DNA was isolated from Methylomonas 16a according tostandard protocols.

[0164] Genomic DNA and library construction were prepared according topublished protocols (Fraser et al The Minimal Gene Complement ofMycoplasma genitalium; Science 270, 1995). A cell pellet was resuspendedin a solution containing 100 mM Na-EDTA pH 8.0, 10 mM tris-HCl pH 8.0,400 mM NaCl, and 50 mM MgCl2.

[0165] Genomic DNA preparation After resuspension, the cells were gentlylysed in 10% SDS, and incubated for 30 minutes at 55° C. Afterincubation at room temperature, proteinase K was added to 100 μg/ml andincubated at 37° C. until the suspension was clear. DNA was extractedtwice with tris-equilibrated phenol and twice with chloroform. DNA wasprecipitated in 70% ethanol and resuspended in a solution containing 10mM tris-HCl and 1 mM Na-EDTA (TE) pH 7.5. The DNA solution was treatedwith a mix of RNAases, then extracted twice with tris-equilibratedphenol and twice with chloroform. This was followed by precipitation inethanol and resuspension in TE.

[0166] Library construction 200 to 500 μg of chromosomal DNA wasresuspended in a solution of 300 mM sodium acetate, 10 mM tris-HCl, 1 mMNa-EDTA, and 30% glycerol, and sheared at 12 psi for 60 sec in anAeromist Downdraft Nebulizer chamber (IBI Medical products, Chicago,Ill.). The DNA was precipitated, resuspended and treated with Bal31nuclease. After size fractionation, a fraction (2.0 kb, or 5.0 kb) wasexcised, cleaned and a two-step ligation procedure was used to produce ahigh titer library with greater than 99% single inserts.

[0167] Sequencing A shotgun sequencing strategy approach was adopted forthe sequencing of the whole microbial genome (Fleischmann, Robert et alWhole-Genome Random sequencing and assembly of Haemophilus influenzae RdScience, 269: 1995).

[0168] Sequence was generated on an ABI Automatic sequencer using dyeterminator technology (U.S. Pat. No. 5,366,860; EP 272007) using acombination of vector and insert-specific primers. Sequence editing wasperformed in either DNAStar (DNA Star Inc.) or the Wisconsin GCG program(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.) and the CONSED package (version 7.0). All sequences representcoverage at least two times in both directions.

Example 3 Identification and Characterization of Bacteria ORF's

[0169] The carbon flux genes isolated from Methylomonas 16a wereidentified by conducting BLAST (Basic Local Alignment Search Tool;Altschul, S. F., et al., (1993) J. Mol. Biol. 215:403-410; see alsowww.ncbi.nim.nih.gov/BLAST/) searches for similarity to sequencescontained in the BLAST “nr” database (comprising all non-redundantGenBank CDS translations, sequences derived from the 3-dimensionalstructure Brookhaven Protein Data Bank, the SWISS-PROT protein sequencedatabase, EMBL, and DDBJ databases). The sequences obtained in Example 1were analyzed for similarity to all publicly available DNA sequencescontained in the “nr” database using the BLASTN algorithm provided bythe National Center for Biotechnology Information (NCBI). The DNAsequences were translated in all reading frames and compared forsimilarity to all publicly available protein sequences contained in the“nr” database using the BLASTX algorithm (Gish, W. and States, D. J.(1993) Nature Genetics 3:266-272) provided by the NCBI. All comparisonswere done using either the BLASTNnr or BLASTXnr algorithm. The resultsof the BLAST comparison is given in Table 1 which summarize thesequences to which they have the most similarity. Table 1 displays databased on the BLASTXnr algorithm with values reported in expect values.The Expect value estimates the statistical significance of the match,specifying the number of matches, with a given score, that are expectedin a search of a database of this size absolutely by chance. TABLE 1Genes Characterized From Methylomonas 16a Similarity SEQ ID % Gene NameIdentified SEQ ID Peptide Identity^(a) Similarity^(b) E-value^(c)Citation Transaldolase Transaldolase 1 2 78% 90% 2.7e-92 Kohler,U., etal., GI:1729831 Plant Mol. Biol. 30 (1), Anabaena 213-218 (1996)variabilis. MIPB - Transaldolase 3 4 50% 79% 1e-23 Blattner F. R. et. altransaldolase GI:7443254 Science 277:1453- Escherichia coli 1474(1997).FBA or FDA Fructose 5 6 76% 92% 4.1e-111 Alefounder P. R. et. al.bisphosphate Mol. Microbiol. 3:723- aldolase 732(1989). FBA or FDAFructose 7 8 40% 70% 2.3e-39 van den Bergh E.R. bisphosphate et al.;aldolase J. Bacteriol. 178:888- 893 (1996). KHG/KDPG (AL352972) 9 10 59%72% 1e-64 Redenbach et al., Mol. Aldolase KHG/KDPG Microbiol. 21 (1),77-96 aldolase (1996) Streptomyces coelicolor Phospho- Phospho- 11 1265% 85% 1.7e-140 Lepek et al., Direct glucomutase glucomutase Submission(Glucose |gb|AAD03475.1| Phosphomutase) (Pgm)>>gi|3241933|gb|AAD03475.1| Glucose 6 Glucose 6 13 14 64% 81% 1.6e-136 Blattner etal., Nucleic phosphate phosphate Acids Res. 21 (23), isomerase isomerase5408-5417 (1993) gi|396360|gb| AAC43119.1 Phosphofruc- Phosphofruc- 1516 63% 83% 1.7e-97 Ladror et al., J. Biol. tokinase tokinase Chem. 266,16550- pyrophosphate pyrophosphate 16555 (1991) dependent dependentgi|150931|gb| AAA25675.1| (M67447) 6- 6- 17 18 60% 85% 1.6e-141 Williset al., J. Bacteriol. Phospho- Phospho- 181 (14), 4176-4184 gluconategluconate (1999) dehydratase dehydratase gi|4210902|gb| AAD12045.1|(AF045609) Glucose 6 Glucose 6 19 20 58% 85% 9.4e-123 Hugouvieux-Cotte-phosphate 1 phosphate 1 Pattat,N, TITLE Direct dehydrogenasedehydrogenase Submission, gi|397854|emb| gi|397854|emb|CAA528CAA52858.1| 58.1| (X74866) (X74866)

[0170]

1 20 1 984 DNA METHYLOMONAS SP. 1 atggcaagaa acttacttga gcaactccgcgagatgaccg ttgttgttgc cgataccggt 60 gacatccagg cgatcgaaac cttcaagccgcgcgatgcaa cgaccaaccc gtctttgatc 120 accgccgcgg cgcaaatgcc gcaatatcaaggcatcgttg acgacacctt gaaaggtgcg 180 cgtgcgacgt tgggtgccag cgcttcggctgccgaggtgg cttcattggc gttcgatcgt 240 ttggcggttt ctttcggttt gaaaatcctggaaatcatcg aaggtcgcgt ttccaccgag 300 gttgatgcgc gtttgtctta tgacaccgaaggcactattg ccaaaggccg ggatctgatc 360 aaacaatacg aagctgcagg tgtttccaaagagcgcgtac tgatcaaaat tgccgcgacc 420 tgggaaggca tccaggcggc tgccgttttggaaaaagaag gtattcacac caacttgacc 480 ctgttgttcg gtctgcacca ggcgattgcttgtgccgaaa acggcattac cctgatttct 540 ccgtttgtcg gccgtattct ggactggtacaaaaaagaca ctggccgcga ctcttatcct 600 tccaacgaag atcctggcgt attgtctgtaactgaagttt ataactacta caaaaaattt 660 ggttataaaa ctgaagtcat gggcgcgagcttccgtaaca tcggcgaaat caccgaattg 720 gcgggttgcg atctgttgac catcgcgccttctctgctgg ccgaactgca atccgttgaa 780 ggtgatttgc cacgcaaact ggaccctgcaaaagcagccg gttcttcgat cgaaaaaatc 840 agcgttgaca aagcgacttt cgagcgcatgcacgaagaaa accgcatggc caaagaaaaa 900 ctggccgaag gtatcgacgg ttttgcgaaagcgttggaaa ccttggaaaa attgttggcg 960 gatcgtttgg ctgctctgga agca 984 2328 PRT METHYLOMONAS SP. 2 Met ala Arg Asn Leu Leu Glu Gln Leu Arg GluMet Thr Val Val Val 1 5 10 15 Ala Asp Thr Gly Asp Ile Gln Ala Ile GluThr Phe Lys Pro Arg Asp 20 25 30 Ala Thr Thr Asn Pro Ser Leu Ile Thr AlaAla Ala Gln Met Pro Gln 35 40 45 Tyr Gln Gly Ile Val Asp Asp Thr Leu LysGly Ala Arg Ala Thr Leu 50 55 60 Gly Ala Ser Ala Ser Ala Ala Glu Val AlaSer Leu Ala Phe Asp Arg 65 70 75 80 Leu Ala Val Ser Phe Gly Leu Lys IleLeu Glu Ile Ile Glu Gly Arg 85 90 95 Val Ser Thr Glu Val Asp Ala Arg LeuSer Tyr Asp Thr Glu Gly Thr 100 105 110 Ile Ala Lys Gly Arg Asp Leu IleLys Gln Tyr Glu Ala Ala Gly Val 115 120 125 Ser Lys Glu Arg Val Leu IleLys Ile Ala Ala Thr Trp Glu Gly Ile 130 135 140 Gln Ala Ala Ala Val LeuGlu Lys Glu Gly Ile His Thr Asn Leu Thr 145 150 155 160 Leu Leu Phe GlyLeu His Gln Ala Ile Ala Cys Ala Glu Asn Gly Ile 165 170 175 Thr Leu IleSer Pro Phe Val Gly Arg Ile Leu Asp Trp Tyr Lys Lys 180 185 190 Asp ThrGly Arg Asp Ser Tyr Pro Ser Asn Glu Asp Pro Gly Val Leu 195 200 205 SerVal Thr Glu Val Tyr Asn Tyr Tyr Lys Lys Phe Gly Tyr Lys Thr 210 215 220Glu Val Met Gly Ala Ser Phe Arg Asn Ile Gly Glu Ile Thr Glu Leu 225 230235 240 Ala Gly Cys Asp Leu Leu Thr Ile Ala Pro Ser Leu Leu Ala Glu Leu245 250 255 Gln Ser Val Glu Gly Asp Leu Pro Arg Lys Leu Asp Pro Ala LysAla 260 265 270 Ala Gly Ser Ser Ile Glu Lys Ile Ser Val Asp Lys Ala ThrPhe Glu 275 280 285 Arg Met His Glu Glu Asn Arg Met ala Lys Glu Lys LeuAla Glu Gly 290 295 300 Ile Asp Gly Phe Ala Lys Ala Leu Glu Thr Leu GluLys Leu Leu Ala 305 310 315 320 Asp Arg Leu Ala Ala Leu Glu Ala 325 3480 DNA METHYLOMONAS SP. 3 atggccgcgg gcggcgtggg cttgacgcaa ttgctgccagaactggccga agctattggt 60 ccgacgagcc gatttcatgt gcaggtcatt ggtgacacggtggaggacat cgttgcggaa 120 gccaaacggc tacacgattt gcccgtcgac atagtggtgaaaattccggc gcatggcgcc 180 ggactggcgg ccatcaagca gatcaagcgc cacgatattccggtgctggc gacagcgatt 240 tacaacgtgc agcaaggttg gctggcggct ttgaacggcgccgattatct ggcgccttat 300 ctgaatcgcg tcgataacca gggttttgac ggtattggcgtggtcgccga tctgcagagc 360 ttgatcgacc ggtatcaaat gcccaccaaa ctcctggtagcgagcttcaa aaacgtacaa 420 caggtgctgc aggtgttgaa actgggcgtg gcgtcggtgacgctgccttt ggacattgtg 480 4 160 PRT METHYLOMONAS SP. 4 Met ala Ala GlyGly Val Gly Leu Thr Gln Leu Leu Pro Glu Leu Ala 1 5 10 15 Glu Ala IleGly Pro Thr Ser Arg Phe His Val Gln Val Ile Gly Asp 20 25 30 Thr Val GluAsp Ile Val Ala Glu Ala Lys Arg Leu His Asp Leu Pro 35 40 45 Val Asp IleVal Val Lys Ile Pro Ala His Gly Ala Gly Leu Ala Ala 50 55 60 Ile Lys GlnIle Lys Arg His Asp Ile Pro Val Leu Ala Thr Ala Ile 65 70 75 80 Tyr AsnVal Gln Gln Gly Trp Leu Ala Ala Leu Asn Gly Ala Asp Tyr 85 90 95 Leu AlaPro Tyr Leu Asn Arg Val Asp Asn Gln Gly Phe Asp Gly Ile 100 105 110 GlyVal Val Ala Asp Leu Gln Ser Leu Ile Asp Arg Tyr Gln Met Pro 115 120 125Thr Lys Leu Leu Val Ala Ser Phe Lys Asn Val Gln Gln Val Leu Gln 130 135140 Val Leu Lys Leu Gly Val Ala Ser Val Thr Leu Pro Leu Asp Ile Val 145150 155 160 5 1005 DNA METHYLOMONAS SP. 5 atggctttag tgtcattgcgacaacttttg gattatgcgg ccgagcatgg ctttgccgtg 60 ccggcgttca acgtcagcaacatggagcag gtacaggcca tcatgcaggc ggccgctgcc 120 tgcgatagtc cagtgatcatgcaaggttcg gccggcgcca accgctatgc cggcgaagtg 180 tttctacggc atttgatattggcggccgtg gagcaatatc cgcatattcc ggtcgtcatg 240 caccgcgacc atgcacccacgcccgacatc tgcgcgcaag ccatacaatc gggcttcagc 300 tcggtgatga tggacggttcgttgctggca gacatgaaaa ccccggcttc ttttgcatac 360 aacgtcgacg tcacccgcaccgtggtcaag atggcgcatg cctgcggcgt atcggtggaa 420 ggcgaaatcg gctgcctgggagcgctggag gccaagtccg cgcaagatca cagccgtttg 480 ctgaccgatc ccgacgaagcggtcgaattc gtcgaacaga cccaggtcga tgccgtggcc 540 gtggccatcg gcaccagccacggcgcctat aaattcagca agccgcccac cggcgaagtg 600 ctggtgatca gtcgattgaaagaactgcag caacgactgc caaataccca ttttgtgatg 660 catggctcca gttcggtgccgcaggattgg ttgaaaatca tcaacgatta tggcggcgat 720 attccggaaa cctatggcgtgccggtcgaa gaaatcgtcg aaggcataaa atatggtgtg 780 cgcaaggtca acatcgataccgacctgcgc atggcgtcca ccggcgcgat gcgcaggttt 840 ctggcccaac cggaaaacgcctcggagcta gacgcgcgca agacctatca agccgccagg 900 gacgcaatgc aggcattatgccaggctcgc tacgaagcgt tcggttcggc gggacatgcc 960 ggcaaaatca aaccggtttcactggcggca atggccaaac gctat 1005 6 335 PRT METHYLOMONAS SP. 6 Met alaLeu Val Ser Leu Arg Gln Leu Leu Asp Tyr Ala Ala Glu His 1 5 10 15 GlyPhe Ala Val Pro Ala Phe Asn Val Ser Asn Met Glu Gln Val Gln 20 25 30 AlaIle Met Gln Ala Ala Ala Ala Cys Asp Ser Pro Val Ile Met Gln 35 40 45 GlySer Ala Gly Ala Asn Arg Tyr Ala Gly Glu Val Phe Leu Arg His 50 55 60 LeuIle Leu Ala Ala Val Glu Gln Tyr Pro His Ile Pro Val Val Met 65 70 75 80His Arg Asp His Ala Pro Thr Pro Asp Ile Cys Ala Gln Ala Ile Gln 85 90 95Ser Gly Phe Ser Ser Val Met Met Asp Gly Ser Leu Leu Ala Asp Met 100 105110 Lys Thr Pro Ala Ser Phe Ala Tyr Asn Val Asp Val Thr Arg Thr Val 115120 125 Val Lys Met ala His Ala Cys Gly Val Ser Val Glu Gly Glu Ile Gly130 135 140 Cys Leu Gly Ala Leu Glu Ala Lys Ser Ala Gln Asp His Ser ArgLeu 145 150 155 160 Leu Thr Asp Pro Asp Glu Ala Val Glu Phe Val Glu GlnThr Gln Val 165 170 175 Asp Ala Val Ala Val Ala Ile Gly Thr Ser His GlyAla Tyr Lys Phe 180 185 190 Ser Lys Pro Pro Thr Gly Glu Val Leu Val IleSer Arg Leu Lys Glu 195 200 205 Leu Gln Gln Arg Leu Pro Asn Thr His PheVal Met His Gly Ser Ser 210 215 220 Ser Val Pro Gln Asp Trp Leu Lys IleIle Asn Asp Tyr Gly Gly Asp 225 230 235 240 Ile Pro Glu Thr Tyr Gly ValPro Val Glu Glu Ile Val Glu Gly Ile 245 250 255 Lys Tyr Gly Val Arg LysVal Asn Ile Asp Thr Asp Leu Arg Met ala 260 265 270 Ser Thr Gly Ala MetArg Arg Phe Leu Ala Gln Pro Glu Asn Ala Ser 275 280 285 Glu Leu Asp AlaArg Lys Thr Tyr Gln Ala Ala Arg Asp Ala Met Gln 290 295 300 Ala Leu CysGln Ala Arg Tyr Glu Ala Phe Gly Ser Ala Gly His Ala 305 310 315 320 GlyLys Ile Lys Pro Val Ser Leu Ala Ala Met ala Lys Arg Tyr 325 330 335 71074 DNA METHYLOMONAS SP. 7 atgacaaaaa tcttagatgt tgtaaaaccc ggcgttgtcaccggtgaaga tgtgcaaaaa 60 attttcgcaa tctgcaaaga aaacaacttt gccttgccagccgtcaacgt gatcagtacc 120 gataccatta atgcggtatt ggaagcggcc gccaaagccaaatcacctgt tgttatccag 180 ttttcaaatg gcggcgcggc tttcgttgcc ggtaaaggtttgaaattgga aggtcaaggc 240 tgttcgattc atggtgccat ttcaggtgct caccacgttcaccgcttggc ggaactctat 300 ggtgtacctg tcgttctgca taccgaccac gcggcgaaaaaattgctgcc atgggtagat 360 ggtatgctgg atgaaggtga aaaattcttt gcggccaccggcaagccttt gttcagctcg 420 cacatgctgg acttgtccga agagagcctg gaagaaaacatcgaaatctg cggtaaatac 480 ttggcgcgca tggcgaaaat gggtatgacc ttggaaatcgaactgggctg caccggcggt 540 gaagaagacg gcgtggacaa cagcggcatg gatcattccgcgttgtacac ccagccggaa 600 gacgtggctt acgcgtatga gcacctgagc aaaatcagccctaacttcac gattgcggct 660 tctttcggca acgtgcacgg cgtttactcg ccaggaaacgtcaagctgac gccaaaaatt 720 ctggataact cgcaaaaata cgtatccgaa aaattcggcttgccagctaa atcattgacc 780 ttcgtattcc atggcggctc tggttcgtct ccggaagaaatcaaggaatc catcagctat 840 ggcgtagtga aaatgaacat cgataccgat acccaatgggcaacctggga aggcgtgatg 900 aacttctaca agaaaaacga aggctatctg caaggccagatcggcaatcc ggaaggtgcc 960 gacaagccga acaaaaaata ctatgaccca cgcgtatggcaacgtgccgg ccaagaaggc 1020 atggttgcac gtctgcaaca agcattccag gaattgaatgcagtaaacac gctg 1074 8 358 PRT METHYLOMONAS SP. 8 Met Thr Lys Ile LeuAsp Val Val Lys Pro Gly Val Val Thr Gly Glu 1 5 10 15 Asp Val Gln LysIle Phe Ala Ile Cys Lys Glu Asn Asn Phe Ala Leu 20 25 30 Pro Ala Val AsnVal Ile Ser Thr Asp Thr Ile Asn Ala Val Leu Glu 35 40 45 Ala Ala Ala LysAla Lys Ser Pro Val Val Ile Gln Phe Ser Asn Gly 50 55 60 Gly Ala Ala PheVal Ala Gly Lys Gly Leu Lys Leu Glu Gly Gln Gly 65 70 75 80 Cys Ser IleHis Gly Ala Ile Ser Gly Ala His His Val His Arg Leu 85 90 95 Ala Glu LeuTyr Gly Val Pro Val Val Leu His Thr Asp His Ala Ala 100 105 110 Lys LysLeu Leu Pro Trp Val Asp Gly Met Leu Asp Glu Gly Glu Lys 115 120 125 PhePhe Ala Ala Thr Gly Lys Pro Leu Phe Ser Ser His Met Leu Asp 130 135 140Leu Ser Glu Glu Ser Leu Glu Glu Asn Ile Glu Ile Cys Gly Lys Tyr 145 150155 160 Leu Ala Arg Met ala Lys Met Gly Met Thr Leu Glu Ile Glu Leu Gly165 170 175 Cys Thr Gly Gly Glu Glu Asp Gly Val Asp Asn Ser Gly Met AspHis 180 185 190 Ser Ala Leu Tyr Thr Gln Pro Glu Asp Val Ala Tyr Ala TyrGlu His 195 200 205 Leu Ser Lys Ile Ser Pro Asn Phe Thr Ile Ala Ala SerPhe Gly Asn 210 215 220 Val His Gly Val Tyr Ser Pro Gly Asn Val Lys LeuThr Pro Lys Ile 225 230 235 240 Leu Asp Asn Ser Gln Lys Tyr Val Ser GluLys Phe Gly Leu Pro Ala 245 250 255 Lys Ser Leu Thr Phe Val Phe His GlyGly Ser Gly Ser Ser Pro Glu 260 265 270 Glu Ile Lys Glu Ser Ile Ser TyrGly Val Val Lys Met Asn Ile Asp 275 280 285 Thr Asp Thr Gln Trp Ala ThrTrp Glu Gly Val Met Asn Phe Tyr Lys 290 295 300 Lys Asn Glu Gly Tyr LeuGln Gly Gln Ile Gly Asn Pro Glu Gly Ala 305 310 315 320 Asp Lys Pro AsnLys Lys Tyr Tyr Asp Pro Arg Val Trp Gln Arg Ala 325 330 335 Gly Gln GluGly Met Val Ala Arg Leu Gln Gln Ala Phe Gln Glu Leu 340 345 350 Asn AlaVal Asn Thr Leu 355 9 636 DNA METHYLOMONAS SP. 9 gaaaatacta tgtccgtcaccatcaaagaa gtcatgacca cctcgcccgt tatgccggtc 60 atggtcatca atcatctggaacatgccgtc cctctggctc gcgcgctagt cgacggtggc 120 ttgaaagttt tggagatcacattgcgcacg ccggtggcac tggaatgtat ccgacgtatc 180 aaagccgaag taccggacgccatcgtcggc gcgggcacca tcatcaaccc tcataccttg 240 tatcaagcga ttgacgccggtgcggaattc atcgtcagcc ccggcatcac cgaaaatcta 300 ctcaacgaag cgctagcatccggcgtgcct atcctgcccg gcgtcatcac acccagcgag 360 gtcatgcgtt tattggaaaaaggcatcaat gcgatgaaat tctttccggc tgaagccgcc 420 ggcggcatac cgatgctgaaatcccttggc ggccccttgc cgcaagtcac cttctgtccg 480 accggcggcg tcaatcccaaaaacgcgccc gaatatctgg cattgaaaaa tgtcgcctgc 540 gtcggcggct cctggatggcgccggccgat ctggtagatg ccgaagactg ggcggaaatc 600 acgcggcggg cgagcgaggccgcggcattg aaaaaa 636 10 212 PRT METHYLOMONAS SP. 10 Glu Asn Thr Met SerVal Thr Ile Lys Glu Val Met Thr Thr Ser Pro 1 5 10 15 Val Met Pro ValMet Val Ile Asn His Leu Glu His Ala Val Pro Leu 20 25 30 Ala Arg Ala LeuVal Asp Gly Gly Leu Lys Val Leu Glu Ile Thr Leu 35 40 45 Arg Thr Pro ValAla Leu Glu Cys Ile Arg Arg Ile Lys Ala Glu Val 50 55 60 Pro Asp Ala IleVal Gly Ala Gly Thr Ile Ile Asn Pro His Thr Leu 65 70 75 80 Tyr Gln AlaIle Asp Ala Gly Ala Glu Phe Ile Val Ser Pro Gly Ile 85 90 95 Thr Glu AsnLeu Leu Asn Glu Ala Leu Ala Ser Gly Val Pro Ile Leu 100 105 110 Pro GlyVal Ile Thr Pro Ser Glu Val Met Arg Leu Leu Glu Lys Gly 115 120 125 IleAsn Ala Met Lys Phe Phe Pro Ala Glu Ala Ala Gly Gly Ile Pro 130 135 140Met Leu Lys Ser Leu Gly Gly Pro Leu Pro Gln Val Thr Phe Cys Pro 145 150155 160 Thr Gly Gly Val Asn Pro Lys Asn Ala Pro Glu Tyr Leu Ala Leu Lys165 170 175 Asn Val Ala Cys Val Gly Gly Ser Trp Met ala Pro Ala Asp LeuVal 180 185 190 Asp Ala Glu Asp Trp Ala Glu Ile Thr Arg Arg Ala Ser GluAla Ala 195 200 205 Ala Leu Lys Lys 210 11 1434 DNA METHYLOMONAS SP. 11aacatgcaaa taaaaaccta taagaccaca ccctatgatg atcaaaaacc cggcacatcc 60gggctaagaa aaaaggttaa agtttttcag caatccggct atctggaaaa tttcgttcag 120tccattttca atagtttaga agattttcag ggaaaaattc tagttttagg cggcgacggc 180cgatatttta atcgacaagc gattcagatc atcatcaaaa tggcggccgc taacgggttt 240ggtgagctga tcatcggcca gggcggtctg ttgtcgacac cggcggcctc caatgtcatc 300cgcaaatatc gcgctttcgg cggcatcatt ctatccgcca gccacaatcc cggtggtccc 360gacgaagact tcggcatcaa atataacgtc ggcaatggcg ggccggcacc ggaaaagttc 420accgacgcct tgttcgaaaa cagcaaaacc atcaccagct atcagatggc cgaaatcgac 480gacatcgatc tcgatagcgt cggcgacgtc caaatcgatg gcataacaat ccgcatcatc 540gatcccgtgg ccgattacgc cgaattgatg gcccggattt tcgatttcga cctgatcaag 600caaagcatcg ccgccggctt gattaccttg cgcttcgacg cgatgcatgc cattaccggc 660ccctatgcca aacatatact cgaagacgtg ctgggcgccg cgcccggttc ggtattcaac 720gccgtaccgc tggaagactt cggcggcggc catcccgatc ccaacatggc gcacgcgcac 780gagctcaccg aaatcatgtt cgggccgaat ccgccggttt tcggcgcggc ctcggacggt 840gacggcgacc gcaacatgat catgggcgcc aatattttcg tcacccccag cgacagtctg 900gccatcatgg cggccaacgc gcaattgatt cccgcctatg ccaagggcat tagcggcgtc 960gcccgctcga tgccgaccag ccaggcggtc gacagggtcg cggataaatt gagtctgccg 1020tgctacgaaa cgccgaccgg ctggaaattc tttggcaatt tgctggatgc cgacaaaatc 1080acgctgtgcg gcgaagaaag cttcggttcc ggttccaatc atgtccggga aaaagacggc 1140ttgtgggccg ttttattttg gctgaatttg cttgcgcgca agcgtcaacc ggccgaggat 1200atcgtgcgtg aacattggca aaaatacggc cgcgacatct attgccgcca tgattacgaa 1260gccgtcgatg ccgacatcgc caacggcatc gtagagcagc tgcgaaacca attgccgagc 1320ttgcccggca aaacctgggg cgattacagc gtcaaattcg ccgacgaatt cagctatacc 1380gatccggtcg atggtagcgt cagcagcaac caaggcatcc gcgtcggttt cgcc 1434 12 545PRT METHYLOMONAS SP. 12 Asn Met Gln Ile Lys Thr Tyr Lys Thr Thr Pro TyrAsp Asp Gln Lys 1 5 10 15 Pro Gly Thr Ser Gly Leu Arg Lys Lys Val LysVal Phe Gln Gln Ser 20 25 30 Gly Tyr Leu Glu Asn Phe Val Gln Ser Ile PheAsn Ser Leu Glu Asp 35 40 45 Phe Gln Gly Lys Ile Leu Val Leu Gly Gly AspGly Arg Tyr Phe Asn 50 55 60 Arg Gln Ala Ile Gln Ile Ile Ile Lys Met alaAla Ala Asn Gly Phe 65 70 75 80 Gly Glu Leu Ile Ile Gly Gln Gly Gly LeuLeu Ser Thr Pro Ala Ala 85 90 95 Ser Asn Val Ile Arg Lys Tyr Arg Ala PheGly Gly Ile Ile Leu Ser 100 105 110 Ala Ser His Asn Pro Gly Gly Pro AspGlu Asp Phe Gly Ile Lys Tyr 115 120 125 Asn Val Gly Asn Gly Gly Pro AlaPro Glu Lys Phe Thr Asp Ala Leu 130 135 140 Phe Glu Asn Ser Lys Thr IleThr Ser Tyr Gln Met ala Glu Ile Asp 145 150 155 160 Asp Ile Asp Leu AspSer Val Gly Asp Val Gln Ile Asp Gly Ile Thr 165 170 175 Ile Arg Ile IleAsp Pro Val Ala Asp Tyr Ala Glu Leu Met ala Arg 180 185 190 Ile Phe AspPhe Asp Leu Ile Lys Gln Ser Ile Ala Ala Gly Leu Ile 195 200 205 Thr LeuArg Phe Asp Ala Met His Ala Ile Thr Gly Pro Tyr Ala Lys 210 215 220 HisIle Leu Glu Asp Val Leu Gly Ala Ala Pro Gly Ser Val Phe Asn 225 230 235240 Ala Val Pro Leu Glu Asp Phe Gly Gly Gly His Pro Asp Pro Asn Met 245250 255 Ala His Ala His Glu Leu Thr Glu Ile Met Phe Gly Pro Asn Pro Pro260 265 270 Val Phe Gly Ala Ala Ser Asp Gly Asp Gly Asp Arg Asn Met IleMet 275 280 285 Gly Ala Asn Ile Phe Val Thr Pro Ser Asp Ser Leu Ala IleMet ala 290 295 300 Ala Asn Ala Gln Leu Ile Pro Ala Tyr Ala Lys Gly IleSer Gly Val 305 310 315 320 Ala Arg Ser Met Pro Thr Ser Gln Ala Val AspArg Val Ala Asp Lys 325 330 335 Leu Ser Leu Pro Cys Tyr Glu Thr Pro ThrGly Trp Lys Phe Phe Gly 340 345 350 Asn Leu Leu Asp Ala Asp Lys Ile ThrLeu Cys Gly Glu Glu Ser Phe 355 360 365 Gly Ser Gly Ser Asn His Val ArgGlu Lys Asp Gly Leu Trp Ala Val 370 375 380 Leu Phe Trp Leu Asn Leu LeuAla Arg Lys Arg Gln Pro Ala Glu Asp 385 390 395 400 Ile Val Arg Glu HisTrp Gln Lys Tyr Gly Arg Asp Ile Tyr Cys Arg 405 410 415 His Asp Tyr GluAla Val Asp Ala Asp Ile Ala Asn Gly Ile Val Glu 420 425 430 Gln Leu ArgAsn Gln Leu Pro Ser Leu Pro Gly Lys Thr Trp Gly Asp 435 440 445 Tyr SerVal Lys Phe Ala Asp Glu Phe Ser Tyr Thr Asp Pro Val Asp 450 455 460 GlySer Val Ser Ser Asn Gln Gly Ile Arg Val Gly Phe Ala Asn Gly 465 470 475480 Ser Arg Ile Val Phe Arg Leu Ser Gly Thr Gly Thr Val Gly Ala Thr 485490 495 Leu Arg Ile Tyr Leu Glu Arg Tyr Glu Arg Asp Val Arg Asn His Asp500 505 510 Gln Asp Pro Gln Val Ala Leu Ala Glu Leu Ile Glu Ile Ala GluGln 515 520 525 Leu Cys Gln Val Lys Gln Arg Thr Gly Arg Thr Glu Pro SerVal Ile 530 535 540 Thr 545 13 1387 DNA METHYLOMONAS SP. 13 ccgaaagcaggcaaaatcac ggttcatttt tttttgtcat ccgtcaaaga caatccttat 60 aatgaggtaatcgttctcct cgctacatct ggcactaaag cttccgaaga ctctttatcc 120 ggttcacacaaaaataatat gtccaaatta atcaactctg ccgaatggaa cgccgtcaaa 180 caacatcatcaagaaattgc tggtaaattt tgcatgaaag aggcttttgc caaagatccc 240 cagcgtttcgataaattctc cgtcaccttt aacgacatat tattagacta ttccaaaaac 300 ctgatcgacgagcgcaccat gcccttgctg atcgcattgg caaagcgggc agacttgcgc 360 gagaaaacggaagcgatgtt ttccggctcc atcatcaaca ccaccgaaaa acgcgcggtt 420 ttgcataccgcgctgcgaaa ccgtagcaat acgcccgttt tcttccgcgg ccaggatgtc 480 atgccggaaatcaacaaggt tctggcaaaa atgcgggttt tcgtggaaca ggtgcgttcg 540 ggccaatggacgggctatag cggcaaggcc attaccgata tcgtcaacat cggcattggc 600 ggctcggatctcggcccgaa aatggtcgac accgccttga cgccgtacgg caaaaacggc 660 ttaaaagcgcatttcgtatc caatgtcgat caaaccgaca tcgtcgaaac cctgaaaccg 720 ctcaatccggaaaccacgct gttcctgatt tcatcgaaaa cgtttaccac gcaggaaacc 780 atgaccaatgcgcgctcggc acgtaactgg ttcatgaatg ccgcgcaaga tcccgcccat 840 atcaagaaacatttcatcgc catttccacc aacgaagaaa tggtcaagga attcggcatc 900 gacccggcgaacatgttcga gttctgggac tgggtcggcg ggcgttattc gctctggtcg 960 gtcatcggcatgtcgatagc tttatatatc ggcatggaca atttcgaaga actgctgatg 1020 ggtgcgcacttggccgacga acatttccgc catgcgccct acgaggaaaa cattccggtc 1080 atcatgggcttgctcggcat ctggtacaac aacttcttcg aagcggaaac ctatgccatt 1140 ttgccgtatgcgcaatcctt gaaatatttt gccgattatt tccagcaagg cgacatggaa 1200 agcaacggcaaaagcgcgac gatcaccggt gaaaaagtcg attacaacac gggccccatc 1260 atctggggacagcccggcac caatggtcag cacgccttct ttcaattgat tcaccaaggc 1320 accaaactggttcccggcga ttttctggcg gccgcgcaaa gtcagtatga tttaccggat 1380 caccacg 138714 592 PRT METHYLOMONAS SP. 14 Pro Lys Ala Gly Lys Ile Thr Val His PhePhe Leu Ser Ser Val Lys 1 5 10 15 Asp Asn Pro Tyr Asn Glu Val Ile ValLeu Leu Ala Thr Ser Gly Thr 20 25 30 Lys Ala Ser Glu Asp Ser Leu Ser GlySer His Lys Asn Asn Met Ser 35 40 45 Lys Leu Ile Asn Ser Ala Glu Trp AsnAla Val Lys Gln His His Gln 50 55 60 Glu Ile Ala Gly Lys Phe Cys Met LysGlu Ala Phe Ala Lys Asp Pro 65 70 75 80 Gln Arg Phe Asp Lys Phe Ser ValThr Phe Asn Asp Ile Leu Leu Asp 85 90 95 Tyr Ser Lys Asn Leu Ile Asp GluArg Thr Met Pro Leu Leu Ile Ala 100 105 110 Leu Ala Lys Arg Ala Asp LeuArg Glu Lys Thr Glu Ala Met Phe Ser 115 120 125 Gly Ser Ile Ile Asn ThrThr Glu Lys Arg Ala Val Leu His Thr Ala 130 135 140 Leu Arg Asn Arg SerAsn Thr Pro Val Phe Phe Arg Gly Gln Asp Val 145 150 155 160 Met Pro GluIle Asn Lys Val Leu Ala Lys Met Arg Val Phe Val Glu 165 170 175 Gln ValArg Ser Gly Gln Trp Thr Gly Tyr Ser Gly Lys Ala Ile Thr 180 185 190 AspIle Val Asn Ile Gly Ile Gly Gly Ser Asp Leu Gly Pro Lys Met 195 200 205Val Asp Thr Ala Leu Thr Pro Tyr Gly Lys Asn Gly Leu Lys Ala His 210 215220 Phe Val Ser Asn Val Asp Gln Thr Asp Ile Val Glu Thr Leu Lys Pro 225230 235 240 Leu Asn Pro Glu Thr Thr Leu Phe Leu Ile Ser Ser Lys Thr PheThr 245 250 255 Thr Gln Glu Thr Met Thr Asn Ala Arg Ser Ala Arg Asn TrpPhe Met 260 265 270 Asn Ala Ala Gln Asp Pro Ala His Ile Lys Lys His PheIle Ala Ile 275 280 285 Ser Thr Asn Glu Glu Met Val Lys Glu Phe Gly IleAsp Pro Ala Asn 290 295 300 Met Phe Glu Phe Trp Asp Trp Val Gly Gly ArgTyr Ser Leu Trp Ser 305 310 315 320 Val Ile Gly Met Ser Ile Ala Leu TyrIle Gly Met Asp Asn Phe Glu 325 330 335 Glu Leu Leu Met Gly Ala His LeuAla Asp Glu His Phe Arg His Ala 340 345 350 Pro Tyr Glu Glu Asn Ile ProVal Ile Met Gly Leu Leu Gly Ile Trp 355 360 365 Tyr Asn Asn Phe Phe GluAla Glu Thr Tyr Ala Ile Leu Pro Tyr Ala 370 375 380 Gln Ser Leu Lys TyrPhe Ala Asp Tyr Phe Gln Gln Gly Asp Met Glu 385 390 395 400 Ser Asn GlyLys Ser Ala Thr Ile Thr Gly Glu Lys Val Asp Tyr Asn 405 410 415 Thr GlyPro Ile Ile Trp Gly Gln Pro Gly Thr Asn Gly Gln His Ala 420 425 430 PhePhe Gln Leu Ile His Gln Gly Thr Lys Leu Val Pro Gly Asp Phe 435 440 445Leu Ala Ala Ala Gln Ser Gln Tyr Asp Leu Pro Asp His His Asp Ile 450 455460 Leu Ile Ser Asn Phe Leu Ala Gln Ala Glu Ala Leu Met Arg Gly Lys 465470 475 480 Thr Glu Glu Glu Val Arg Gln Asp Leu Ser His Glu Pro Asn LeuAsp 485 490 495 Asp Ala Leu Ile Ala Ser Lys Ile Phe Glu Gly Asn Lys ProSer Asn 500 505 510 Ser Phe Leu Phe Lys Lys Leu Thr Pro Arg Thr Leu GlyThr Leu Ile 515 520 525 Ala Phe Tyr Glu His Lys Ile Phe Val Gln Gly ValIle Trp Asn Ile 530 535 540 Asn Ser Phe Asp Gln Met Gly Val Glu Leu GlyLys Val Leu Ala Lys 545 550 555 560 Ala Ile Leu Pro Glu Leu Lys Asn AspAsp Ile Ile Ala Ser His Asp 565 570 575 Ser Ser Thr Asn Gly Leu Ile AsnThr Tyr Lys Arg Leu Arg Lys Ala 580 585 590 15 1311 DNA METHYLOMONAS SP.15 gatgtggtca catggcccta tcacttaacg gctgatattc gattttgtca ttggtttttt 60cttaacttta acttctacac gctcatgaac aaacctaaaa aagttgcaat actgacagca 120ggcggcttgg cgccttgttt gaattccgca atcggtagtt tgatcgaacg ttataccgaa 180atcgatccta gcatagaaat catttgctat cgcggcggtt ataaaggcct gttgctgggc 240gattcttatc cagtaacggc cgaagtgcgt aaaaaggcgg gtgttctgca acgttttggc 300ggttctgtga tcggcaacag ccgcgtcaaa ttgaccaatg tcaaagactg cgtgaaacgc 360ggtttggtca aagagggtga agatccgcaa aaagtcgcgg ctgatcaatt ggttaaggat 420ggtgtcgata ttctgcacac catcggcggc gatgatacca atacggcagc agcggatttg 480gcagcattcc tggccagaaa taattacgga ctgaccgtca ttggtttacc taaaaccgtc 540gataacgacg tatttccgat caagcaatca ctaggtgctt ggactgccgc cgagcaaggc 600gcgcgttatt tcatgaacgt ggtggccgaa aacaacgcca acccacgcat gctgatcgta 660cacgaagtga tgggccgtaa ctgcggctgg ctgaccgctg caaccgcgca ggaatatcgc 720aaattactgg accgtgccga gtggttgccg gaattgggtt tgactcgtga atcttatgaa 780gtgcacgcgg tattcgttcc ggaaatggcg atcgacctgg aagccgaagc caagcgcctg 840cgcgaagtga tggacaaagt cgattgcgtc aacatcttcg tttccgaagg tgccggcgtc 900gaagctatcg tcgcggaaat gcaggccaaa ggccaggaag tgccgcgcga tgcgttcggc 960cacatcaaac tggatgcggt caaccctggt aaatggttcg gcgagcaatt cgcgcagatg 1020ataggcgcgg aaaaaaccct ggtacaaaaa tcgggatact tcgcccgtgc ttctgcttcc 1080aacgttgacg acatgcgttt gatcaaatcg tgcgccgact tggcggtcga gtgcgcgttc 1140cgccgcgagt ctggcgtgat cggtcacgac gaagacaacg gcaacgtgtt gcgtgcgatc 1200gagtttccgc gcatcaaggg cggcaaaccg ttcaatatcg acaccgactg gttcaatagc 1260atgttgagcg aaatcggcca gcctaaaggc ggtaaagtcg aagtcagcca c 1311 16 437 PRTMETHYLOMONAS SP. 16 Asp Val Val Thr Trp Pro Tyr His Leu Thr Ala Asp IleArg Phe Cys 1 5 10 15 His Trp Phe Phe Leu Asn Phe Asn Phe Tyr Thr LeuMet Asn Lys Pro 20 25 30 Lys Lys Val Ala Ile Leu Thr Ala Gly Gly Leu AlaPro Cys Leu Asn 35 40 45 Ser Ala Ile Gly Ser Leu Ile Glu Arg Tyr Thr GluIle Asp Pro Ser 50 55 60 Ile Glu Ile Ile Cys Tyr Arg Gly Gly Tyr Lys GlyLeu Leu Leu Gly 65 70 75 80 Asp Ser Tyr Pro Val Thr Ala Glu Val Arg LysLys Ala Gly Val Leu 85 90 95 Gln Arg Phe Gly Gly Ser Val Ile Gly Asn SerArg Val Lys Leu Thr 100 105 110 Asn Val Lys Asp Cys Val Lys Arg Gly LeuVal Lys Glu Gly Glu Asp 115 120 125 Pro Gln Lys Val Ala Ala Asp Gln LeuVal Lys Asp Gly Val Asp Ile 130 135 140 Leu His Thr Ile Gly Gly Asp AspThr Asn Thr Ala Ala Ala Asp Leu 145 150 155 160 Ala Ala Phe Leu Ala ArgAsn Asn Tyr Gly Leu Thr Val Ile Gly Leu 165 170 175 Pro Lys Thr Val AspAsn Asp Val Phe Pro Ile Lys Gln Ser Leu Gly 180 185 190 Ala Trp Thr AlaAla Glu Gln Gly Ala Arg Tyr Phe Met Asn Val Val 195 200 205 Ala Glu AsnAsn Ala Asn Pro Arg Met Leu Ile Val His Glu Val Met 210 215 220 Gly ArgAsn Cys Gly Trp Leu Thr Ala Ala Thr Ala Gln Glu Tyr Arg 225 230 235 240Lys Leu Leu Asp Arg Ala Glu Trp Leu Pro Glu Leu Gly Leu Thr Arg 245 250255 Glu Ser Tyr Glu Val His Ala Val Phe Val Pro Glu Met ala Ile Asp 260265 270 Leu Glu Ala Glu Ala Lys Arg Leu Arg Glu Val Met Asp Lys Val Asp275 280 285 Cys Val Asn Ile Phe Val Ser Glu Gly Ala Gly Val Glu Ala IleVal 290 295 300 Ala Glu Met Gln Ala Lys Gly Gln Glu Val Pro Arg Asp AlaPhe Gly 305 310 315 320 His Ile Lys Leu Asp Ala Val Asn Pro Gly Lys TrpPhe Gly Glu Gln 325 330 335 Phe Ala Gln Met Ile Gly Ala Glu Lys Thr LeuVal Gln Lys Ser Gly 340 345 350 Tyr Phe Ala Arg Ala Ser Ala Ser Asn ValAsp Asp Met Arg Leu Ile 355 360 365 Lys Ser Cys Ala Asp Leu Ala Val GluCys Ala Phe Arg Arg Glu Ser 370 375 380 Gly Val Ile Gly His Asp Glu AspAsn Gly Asn Val Leu Arg Ala Ile 385 390 395 400 Glu Phe Pro Arg Ile LysGly Gly Lys Pro Phe Asn Ile Asp Thr Asp 405 410 415 Trp Phe Asn Ser MetLeu Ser Glu Ile Gly Gln Pro Lys Gly Gly Lys 420 425 430 Val Glu Val SerHis 435 17 1360 DNA METHYLOMONAS SP. 17 agtgtcccgc actcgcatca cccggagacatccttaatgc atcccgtact cgaaaaagtc 60 acagaacaag tcatcgcccg cagccgggaaacccgtgccg cttatctgaa gcgcatagag 120 gccgccatcg ccgaaggccc gcaacgcaataaactgcctt gcgccaatct ggcccacggt 180 ttcgcggtct gttcggccat cgaaaaagaagaattgtctc atggccccaa gcccaatgtc 240 ggcatcatct cggcctacaa cgacatgctgtccgcgcacg aaccctacaa ggattatcct 300 gccctgatca aacaggccgt gcgcgaagccggcggcgtgg ctcaattcgc cggcggcgtg 360 cccgcgatgt gcgacggcgt cacccagggcatgccgggca tggaattgtc gctattcagc 420 cgcgacgtca tcgcgatgtc caccgcgatcggcctggctc ataacatgtt cgacgcggcg 480 ctgtatctgg gcgtctgcga caagatcgtacccggtttgt tgatcggtgc attgagcttc 540 ggccatttgc cggccgtttt cttgccagccggccccatga ccagcggcct gtccaacaag 600 gaaaaatccc gtgcccggca aaaatacgccgaaggtaaga tcggtgaaaa agaattgctg 660 gaatcggaag ccaagtctta ccacagcccaggcacctgca ccttctatgg caccgccaac 720 agcaaccaga tgatggtcga gatcatgggcctgcacctgc ccggtagttc cttcatcaat 780 ccttacaccc cactgcgcga cgaactgaccaaggccgccg ccaggcaggt gttgaaattc 840 accgcgctgg gcaacgactt caggccaatcgcgcatgtga tcgacgaaaa agccatcatc 900 aatgccatca tcggcttgct ggcgaccggcggttcgacca accataccat ccatttgatc 960 gcgattgccc gcgccgccgg catcatcatcaactgggacg atttcgacgc cctatccaaa 1020 gtcattccgt tgctgaccaa gatctatccgaacggcccgg ccgacgtcaa ccaattccag 1080 gcggccggcg gcatgagctt attgatacacgaactgctgg atcacggctt gttgcacggc 1140 gacatcctga ccataggcga ccagcgcggcatggcccaat acagtcaagt accgacgctg 1200 caagacggcc aattacaatg gcagcccggtcctaccgcat cgcgcgatcc cgaaatcatc 1260 gccagcgtgg caaaaccttt cgccgccggtggtggcctgc atgtgatgca tggcaatctg 1320 ggccgcggcg tatccaagat ttccgccgtctccgaagatc 1360 18 618 PRT METHYLOMONAS SP. 18 Ser Val Pro His Ser HisHis Pro Glu Thr Ser Leu Met His Pro Val 1 5 10 15 Leu Glu Lys Val ThrGlu Gln Val Ile Ala Arg Ser Arg Glu Thr Arg 20 25 30 Ala Ala Tyr Leu LysArg Ile Glu Ala Ala Ile Ala Glu Gly Pro Gln 35 40 45 Arg Asn Lys Leu ProCys Ala Asn Leu Ala His Gly Phe Ala Val Cys 50 55 60 Ser Ala Ile Glu LysGlu Glu Leu Ser His Gly Pro Lys Pro Asn Val 65 70 75 80 Gly Ile Ile SerAla Tyr Asn Asp Met Leu Ser Ala His Glu Pro Tyr 85 90 95 Lys Asp Tyr ProAla Leu Ile Lys Gln Ala Val Arg Glu Ala Gly Gly 100 105 110 Val Ala GlnPhe Ala Gly Gly Val Pro Ala Met Cys Asp Gly Val Thr 115 120 125 Gln GlyMet Pro Gly Met Glu Leu Ser Leu Phe Ser Arg Asp Val Ile 130 135 140 AlaMet Ser Thr Ala Ile Gly Leu Ala His Asn Met Phe Asp Ala Ala 145 150 155160 Leu Tyr Leu Gly Val Cys Asp Lys Ile Val Pro Gly Leu Leu Ile Gly 165170 175 Ala Leu Ser Phe Gly His Leu Pro Ala Val Phe Leu Pro Ala Gly Pro180 185 190 Met Thr Ser Gly Leu Ser Asn Lys Glu Lys Ser Arg Ala Arg GlnLys 195 200 205 Tyr Ala Glu Gly Lys Ile Gly Glu Lys Glu Leu Leu Glu SerGlu Ala 210 215 220 Lys Ser Tyr His Ser Pro Gly Thr Cys Thr Phe Tyr GlyThr Ala Asn 225 230 235 240 Ser Asn Gln Met Met Val Glu Ile Met Gly LeuHis Leu Pro Gly Ser 245 250 255 Ser Phe Ile Asn Pro Tyr Thr Pro Leu ArgAsp Glu Leu Thr Lys Ala 260 265 270 Ala Ala Arg Gln Val Leu Lys Phe ThrAla Leu Gly Asn Asp Phe Arg 275 280 285 Pro Ile Ala His Val Ile Asp GluLys Ala Ile Ile Asn Ala Ile Ile 290 295 300 Gly Leu Leu Ala Thr Gly GlySer Thr Asn His Thr Ile His Leu Ile 305 310 315 320 Ala Ile Ala Arg AlaAla Gly Ile Ile Ile Asn Trp Asp Asp Phe Asp 325 330 335 Ala Leu Ser LysVal Ile Pro Leu Leu Thr Lys Ile Tyr Pro Asn Gly 340 345 350 Pro Ala AspVal Asn Gln Phe Gln Ala Ala Gly Gly Met Ser Leu Leu 355 360 365 Ile HisGlu Leu Leu Asp His Gly Leu Leu His Gly Asp Ile Leu Thr 370 375 380 IleGly Asp Gln Arg Gly Met ala Gln Tyr Ser Gln Val Pro Thr Leu 385 390 395400 Gln Asp Gly Gln Leu Gln Trp Gln Pro Gly Pro Thr Ala Ser Arg Asp 405410 415 Pro Glu Ile Ile Ala Ser Val Ala Lys Pro Phe Ala Ala Gly Gly Gly420 425 430 Leu His Val Met His Gly Asn Leu Gly Arg Gly Val Ser Lys IleSer 435 440 445 Ala Val Ser Glu Asp His Gln Val Val Thr Ala Pro Ala MetVal Phe 450 455 460 Asp Asp Gln Leu Asp Val Val Ala Ala Phe Lys Arg GlyGlu Leu Glu 465 470 475 480 Lys Asp Val Ile Val Val Leu Arg Phe Gln GlyPro Lys Ala Asn Gly 485 490 495 Met Pro Glu Leu His Lys Leu Thr Pro ValLeu Gly Val Leu Gln Asp 500 505 510 Arg Gly Phe Lys Val Gly Leu Leu ThrAsp Gly Arg Met Ser Gly Ala 515 520 525 Ser Gly Lys Val Pro Ser Ala IleHis Met Trp Pro Glu Cys Ile Asp 530 535 540 Gly Gly Pro Leu Ala Lys ValArg Asp Gly Asp Ile Ile Val Met Asn 545 550 555 560 Thr Gln Thr Gly GluVal Asn Val Gln Val Asp Pro Ala Glu Phe Lys 565 570 575 Ala Arg Val AlaGlu Pro Asn His Ala Thr Gly His His Phe Gly Met 580 585 590 Gly Arg GluLeu Phe Gly Ala Met Arg Ala Gln Ala Ser Thr Ala Glu 595 600 605 Thr GlyAla Thr Asn Leu Phe Phe Val Asp 610 615 19 1477 DNA METHYLOMONAS SP. 19atggcattgg gctttttgct ccgtagcccc aaagacatga caaaaaacat tacttacaaa 60ccctgcgacc tggtgattta cggcgcactg ggcgatttat ccaaacgtaa actactgatt 120tcattatacc gtttggaaaa acacaatctg ctcgagcccg atacgcgcat catcggcgta 180gatcgtttgg aagaaaccag cgacagtttc gtcgaaattg cgcacaaaag cttgcaggcg 240tttttgaaca acgtcatcga cgcagaaatc tggcaacgtt tttccaaacg cttgtcctat 300ttgaaaatcg atctgaccca acccgagcaa tacaaacaac tgcatacggt cgtcgatgcc 360gaaaaacgag tcatggtgaa ttatttcgcg gtggcaccct ttttgttcaa aaacatttgc 420caaggcttgc atgactgcgg cgtattgacg gccgaatcgc gcatggtgat ggaaaaaccc 480atcggccacg acctgaaatc gtcgaaagaa atcaacgacg tcgtcgccga cgtattccac 540gaagaccagg tctaccgcat cgaccactac ctgggcaagg aaacggtctt gaacttgctg 600gccttgcgtt tcgccaattc gatattcacg accaactgga atcacaacac gatagaccat 660atccagatta cggtcggtga ggacatcggc atcgagggcc gttgggaata tttcgacaag 720accggccaat tgcgcgacat gctgcaaaac catttgctgc aaatcctgac cttcgtcgcg 780atggagccgc ccgcggatct gtcggccgaa agcatacaca tggaaaaaat caaggtcctg 840aaagccttgc ggccaatcac cgtgcgcaat gtcgaggaaa aaaccgtgcg cggtcaatac 900accgccggtt tcatcaaagg caagtcggta ccgggttatc tggaagaaga aggtgccaac 960accgaaagca cgaccgaaac tttcgtcgcg atccgcgtgg atatcgataa ctggcgctgg 1020gccggtgtcc cgttttacat gcgtaccggc aaacgcacgc ccaacaaacg caccgagatt 1080gtggtcaatt tcaagcaatt gccgcacaac atcttcaagg acagttttca tgaactgccg 1140gccaataaac tggtcattca tttgcaaccg aacgaagggg tggatgtcat gatgttgaac 1200aaggtgccgg gcatagacgg caacatcaag ttgcaacaga ccaaactgga tttgagcttt 1260tccgaaacct tcaagaaaaa ccgaattttc ggcggctacg aaaaactgat tctggaagcc 1320ctgcgcggca acccgacgct gtttttgagc cgcgaggaaa tagaacaagc ctggacctgg 1380gtcgattcga ttcaggatgc ctggcaacac aaccacacgc cacccaaacc ctatcccgcg 1440ggtagctggg gtccagtggc atcggtcgca ttactgg 1477 20 501 PRT METHYLOMONASSP. 20 Met ala Leu Gly Phe Leu Leu Arg Ser Pro Lys Asp Met Thr Lys Asn 15 10 15 Ile Thr Tyr Lys Pro Cys Asp Leu Val Ile Tyr Gly Ala Leu Gly Asp20 25 30 Leu Ser Lys Arg Lys Leu Leu Ile Ser Leu Tyr Arg Leu Glu Lys His35 40 45 Asn Leu Leu Glu Pro Asp Thr Arg Ile Ile Gly Val Asp Arg Leu Glu50 55 60 Glu Thr Ser Asp Ser Phe Val Glu Ile Ala His Lys Ser Leu Gln Ala65 70 75 80 Phe Leu Asn Asn Val Ile Asp Ala Glu Ile Trp Gln Arg Phe SerLys 85 90 95 Arg Leu Ser Tyr Leu Lys Ile Asp Leu Thr Gln Pro Glu Gln TyrLys 100 105 110 Gln Leu His Thr Val Val Asp Ala Glu Lys Arg Val Met ValAsn Tyr 115 120 125 Phe Ala Val Ala Pro Phe Leu Phe Lys Asn Ile Cys GlnGly Leu His 130 135 140 Asp Cys Gly Val Leu Thr Ala Glu Ser Arg Met ValMet Glu Lys Pro 145 150 155 160 Ile Gly His Asp Leu Lys Ser Ser Lys GluIle Asn Asp Val Val Ala 165 170 175 Asp Val Phe His Glu Asp Gln Val TyrArg Ile Asp His Tyr Leu Gly 180 185 190 Lys Glu Thr Val Leu Asn Leu LeuAla Leu Arg Phe Ala Asn Ser Ile 195 200 205 Phe Thr Thr Asn Trp Asn HisAsn Thr Ile Asp His Ile Gln Ile Thr 210 215 220 Val Gly Glu Asp Ile GlyIle Glu Gly Arg Trp Glu Tyr Phe Asp Lys 225 230 235 240 Thr Gly Gln LeuArg Asp Met Leu Gln Asn His Leu Leu Gln Ile Leu 245 250 255 Thr Phe ValAla Met Glu Pro Pro Ala Asp Leu Ser Ala Glu Ser Ile 260 265 270 His MetGlu Lys Ile Lys Val Leu Lys Ala Leu Arg Pro Ile Thr Val 275 280 285 ArgAsn Val Glu Glu Lys Thr Val Arg Gly Gln Tyr Thr Ala Gly Phe 290 295 300Ile Lys Gly Lys Ser Val Pro Gly Tyr Leu Glu Glu Glu Gly Ala Asn 305 310315 320 Thr Glu Ser Thr Thr Glu Thr Phe Val Ala Ile Arg Val Asp Ile Asp325 330 335 Asn Trp Arg Trp Ala Gly Val Pro Phe Tyr Met Arg Thr Gly LysArg 340 345 350 Thr Pro Asn Lys Arg Thr Glu Ile Val Val Asn Phe Lys GlnLeu Pro 355 360 365 His Asn Ile Phe Lys Asp Ser Phe His Glu Leu Pro AlaAsn Lys Leu 370 375 380 Val Ile His Leu Gln Pro Asn Glu Gly Val Asp ValMet Met Leu Asn 385 390 395 400 Lys Val Pro Gly Ile Asp Gly Asn Ile LysLeu Gln Gln Thr Lys Leu 405 410 415 Asp Leu Ser Phe Ser Glu Thr Phe LysLys Asn Arg Ile Phe Gly Gly 420 425 430 Tyr Glu Lys Leu Ile Leu Glu AlaLeu Arg Gly Asn Pro Thr Leu Phe 435 440 445 Leu Ser Arg Glu Glu Ile GluGln Ala Trp Thr Trp Val Asp Ser Ile 450 455 460 Gln Asp Ala Trp Gln HisAsn His Thr Pro Pro Lys Pro Tyr Pro Ala 465 470 475 480 Gly Ser Trp GlyPro Val Ala Ser Val Ala Leu Leu Ala Arg Asp Gly 485 490 495 Arg Ala TrpGlu Glu 500

What is claimed is:
 1. An isolated nucleic acid molecule encoding aMethylomonas sp carbon flux enzyme, selected from the group consistingof: (a) an isolated nucleic acid molecule encoding the amino acidsequence selected from the group consisting of SEQ ID NOs:2, 4, 6, 8,10, 12, 14, 16, 18, and 20; (b) an isolated nucleic acid molecule thathybridizes with (a) under the following hybridization conditions:0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS followed by0.1×SSC, 0.1% SDS; and (c) an isolated nucleic acid molecule that iscomplementary to (a) or (b).
 2. The isolated nucleic acid molecule ofclaim 1 selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9,11, 13, 15, 17, and
 19. 3. A polypeptide encoded by the isolated nucleicacid molecule of claim
 1. 4. The polypeptide of claim 3 selected fromthe group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and20.
 5. An isolated nucleic acid molecule comprising a first nucleotidesequence encoding a polypeptide of at least 328 amino acids that has atleast 78% identity based on the Smith-Waterman method of alignment whencompared to a polypeptide having the sequence as set forth in SEQ IDNO:2; or a second nucleotide sequence comprising the complement of thefirst nucleotide sequence.
 6. An isolated nucleic acid moleculecomprising a first nucleotide sequence encoding a polypeptide of atleast 160 amino acids that has at least 50% identity based on theSmith-Waterman method of alignment when compared to a polypeptide havingthe sequence as set forth in SEQ ID NO:4, or a second nucleotidesequence comprising the complement of the first nucleotide sequence. 7.An isolated nucleic acid molecule comprising a first nucleotide sequenceencoding a polypeptide of at least 335 amino acids that has at least 76%identity based on the Smith-Waterman method of alignment when comparedto a polypeptide having the sequence as set forth in SEQ ID NO:6, or asecond nucleotide sequence comprising the complement of the firstnucleotide sequence.
 8. An isolated nucleic acid molecule comprising afirst nucleotide sequence encoding a polypeptide of at least 358 aminoacids that has at least 40% identity based on the Smith-Waterman methodof alignment when compared to a polypeptide having the sequence as setforth in SEQ ID NO:8, or a second nucleotide sequence comprising thecomplement of the first nucleotide sequence.
 9. An isolated nucleic acidmolecule comprising a first nucleotide sequence encoding a polypeptideof at least 212 amino acids that has at least 59% identity based on theSmith-Waterman method of alignment when compared to a polypeptide havingthe sequence as set forth in SEQ ID NO:10, or a second nucleotidesequence comprising the complement of the first nucleotide sequence. 10.An isolated nucleic acid molecule comprising a first nucleotide sequenceencoding a polypeptide of at least 545 amino acids that has at least 65%identity based on the Smith-Waterman method of alignment when comparedto a polypeptide having the sequence as set forth in SEQ ID NO:12, or asecond nucleotide sequence comprising the complement of the firstnucleotide sequence.
 11. An isolated nucleic acid molecule comprising afirst nucleotide sequence encoding a polypeptide of at least 592 aminoacids that has at least 64% identity based on the Smith-Waterman methodof alignment when compared to a polypeptide having the sequence as setforth in SEQ ID NO:14, or a second nucleotide sequence comprising thecomplement of the first nucleotide sequence.
 12. An isolated nucleicacid molecule comprising a first nucleotide sequence encoding apolypeptide of at least 437 amino acids that has at least 63% identitybased on the Smith-Waterman method of alignment when compared to apolypeptide having the sequence as set forth in SEQ ID NO:16, or asecond nucleotide sequence comprising the complement of the firstnucleotide sequence.
 13. An isolated nucleic acid molecule comprising afirst nucleotide sequence encoding a polypeptide of at least 618 aminoacids that has at least 60% identity based on the Smith-Waterman methodof alignment when compared to a polypeptide having the sequence as setforth in SEQ ID NO:18, or a second nucleotide sequence comprising thecomplement of the first nucleotide sequence.
 14. An isolated nucleicacid molecule comprising a first nucleotide sequence encoding apolypeptide of at least 501 amino acids that has at least 58% identitybased on the Smith-Waterman method of alignment when compared to apolypeptide having the sequence as set forth in SEQ ID NO:20, or asecond nucleotide sequence comprising the complement of the firstnucleotide sequence.
 15. A chimeric gene comprising the isolated nucleicacid fragment of claim 1 operably linked to suitable regulatorysequences.
 16. A transformed host cell comprising a host cell and thechimeric gene of claim
 15. 17. The transformed host cell of claim 6wherein the host cell is selected from the group consisting of bacteria,yeast, and filamentous fungi.
 18. The transformed host cell of claim 17wherein the host cell is selected from the group consisting ofAspergillus, Saccharomyces, Pichia, Candida, Hansenula, Salmonella,Bacillus, Acinetobacter, Rhodococcus, Streptomyces, Escherichia,Pseudomonas, Methylomonas, Methylococcs and Methylobacter.
 19. A methodof obtaining a nucleic acid fragment encoding a carbon flux enzymecomprising: (a) probing a genomic library with the nucleic acid fragmentof claim 1; (b) identifying a DNA clone that hybridizes with the nucleicacid fragment of claim 1; and (c) sequencing the genomic fragment thatcomprises the clone identified in step (b), wherein the sequencedgenomic fragment encodes a carbon flux enzyme.
 20. A method of obtaininga nucleic acid fragment encoding a carbon flux enzyme comprising: (a)synthesizing at least one oligonucleotide primer corresponding to aportion of the sequence selected from the group consisting of SEQ IDNO:1, 3, 5, 7, 9, 11, 13, 15, 17, and 19; (b) amplifying an insertpresent in a cloning vector using the oligonucleotide primer of step(a); wherein the amplified insert encodes a portion of an amino acidsequence encoding a carbon flux enzyme.
 21. The product of the method ofclaims 19 or
 20. 22. A method of altering carbon flow through amethanotrophic bacteria comprising, over-expressing at least one carbonflux gene selected from the group consisting of SEQ ID NOs:1, 3, 5, 7,9, 11, 13, 15, 17 and 19 in a methanotrophic strain such that the carbonflow is altered through the strain.
 23. A method according to claim 22wherein said methanotrophic bacteria: (a) grows on a C1 carbon substrateselected from the group consisting of methane and methanol; and (b)comprises a functional Embden-Meyerhof carbon pathway, said pathwaycomprising a gene encoding a pyrophosphate dependent phosphofructokinaseenzyme.
 24. A method according to claim 23 wherein said methanotrophicbacteria is Methylomonas 16a ATCC PTA
 2402. 25. A method according toclaim 22 wherein said carbon flux gene is over-expressed on a multicopyplasmid.
 26. A method according to claim 22 wherein said carbon fluxgene is operably linked to an inducible or regulated promoter.
 27. Amethod according to claim 22 wherein said carbon flux gene is expressedin antisense orientation.
 28. A method according to claim 22 whereinsaid carbon flux gene is disrupted by insertion of foreign DNA into thecoding region.
 29. A mutated gene encoding a carbon flux enzyme havingan altered biological activity produced by a method comprising the stepsof: (i) digesting a mixture of nucleotide sequences with restrictionendonucleases wherein said mixture comprises: a) a native carbon fluxgene; b) a first population of nucleotide fragments which will hybridizeto said native carbon flux gene; c) a second population of nucleotidefragments which will not hybridize to said native carbon flux gene;wherein a mixture of restriction fragments are produced; (ii) denaturingsaid mixture of restriction fragments; (iii) incubating the denaturedsaid mixture of restriction fragments of step (ii) with a polymerase;(iv) repeating steps (ii) and (iii) wherein a mutated carbon flux geneis produced encoding a protein having an altered biological activity.